Maternal consumption of HFD during critical developmental periods has been shown to lead to commitments on offspring's homeostasis and may result in metabolic disorders [11, 28]. We recently showed that maternal consumption of HFD during gestation and lactation activates pro-inflammatory pathways and unfolded protein response, impairs cholinergic anti-inflammatory pathway, modulates autophagy proteins and also affects lipid metabolism in offspring [25, 36–38]. Offspring from HFD-fed dams present downregulation of hepatic β-oxidation-related genes and upregulation of genes involved in lipid synthesis, and these alterations seem to be driven by the modulation of miR-122 and miR-370 levels, causing metabolic adaptations that lead to increased ectopic lipid accumulation in the liver of recently weaned mice .
Despite some studies have suggested that maternal macronutrients may be responsible for gene expression modulation in foetal offspring [39–41], we have not found studies showing that components of maternal diet could directly alter miRNAs expression in offspring. It is known that a HFD consumption increases lipids levels in serum, such as TAG, CHOL and free fatty acids (FFA)  and, moreover, maternal diet can directly affect breast milk composition [32–34, 42]. Therefore we hypothesized that the lipids consumed by the dams, specially SFA, could be responsible for miR-122 and miR-370 modulation in the liver of offspring through placental delivery during gestation and/or milk composition during suckling period.
As shown in the present study, treatment with palmitic acid, one of the most abundant SFAs in the human diet and blood, leads to a decrease in miR-122 and increased miR-370 levels in hepatocytes. It have been largely reported that SFAs play an important role in the development of insulin resistance by their directly regulation of inflammatory and metabolic pathways [15, 16, 43]. More recently, studies showed that several miRNAs are dysregulated in rodent models of diet-induced obesity, type 2 diabetes and NAFLD , reinforcing that excessive fat consumption, particularly SFAs, may be involved in miRNAs modulation that participate in the genesis of metabolic diseases. Importantly, in vitro analysis showed that miRNAs can be direct targets of dietary components, including fatty acids [19, 45]. Moreover, Nie and colleagues (2014) induced lipid accumulation in human hepatocyte cell line using oleic acid. In this steatotic hepatocyte model, miR-122 expression was downregulated and, importantly, transfection with miR-122 mimic significantly reduced lipids within the hepatocytes .
After confirming that the in vitro treatment with palmitate was able to reduce miR-122 and increase miR-370 expression, we aimed to investigate the independent contribution of maternal excessive lipids consumption at gestational or lactation periods on the modulation of these miRNAs, since only few studies have investigated the relative contribution of pre- or post-natal maternal HFD consumption to metabolic phenotype in offspring. Sun and colleagues  showed that maternal HFD consumption during the suckling period has a high influence on leptin signaling in offspring. On the other hand, Cerf and colleagues  showed that neonates exposed to HFD only during foetal development presented reduced volume and number of β-cells, accompanied by sustained and irreversible hyperglycaemia during adulthood.
Here, we showed that pups from HFD-fed dams presents lower body weight and LIO at birth (d0), in comparison to pups from control dams. Similar studies have shown higher body weight and macrosomia in newborns from obese dams [49, 50] however, some authors have shown reduced body weight in offspring at d0 using models of maternal hyperglycemia and transient hyperinsulinemia. In our model, HFD-fed dams presented higher insulin levels prior to mating when compared to control dams and, although this hormone does not cross the placental barrier, some authors suggest that maternal hyperinsulinemia may lead to placental disorders and that this occurrence may be related to the delayed foetus development [51–53].
Interestingly, besides the lower body weight, hepatic lipid synthesis seems to be increased in offspring from HFD-fed dams at birth, as suggested by liver Agpat and Gpam levels and, in contrast, genes related to fatty acid oxidation (Cpt1a and Acadvl) are reduced in their liver. There are several evidences in humans indicating that maternal obesity leads to increased fuel availability for the foetus and may drive to increased hepatic fat storage . Maternal nutritional overload and placental transfer is a challenge to foetal development and, as suggested by Brumbaugh and Friedman , until final gestational phases, subcutaneous fat is not available to act as a storage buffer so, under dietary excesses, the foetal liver, among another organs, becomes a fat deposit. McCurdy and co-workers  showed, in non-human primates, an increase in hepatic TAG and elevated hepatic expression of gluconeogenesis key genes in foetus from HFD-fed dams at gestational third trimester and the maternal resistance to obesity development did not reduce the effects of the HFD-consumption on hepatic metabolism of the fetuses. Therefore, these findings suggest that maternal HFD consumption during pregnancy can modulate lipid metabolism in offspring, favoring fat deposition.
As we have speculated, miR-122 and miR-370 may be involved in the impairments of lipid homeostasis of newborns from HFD-fed dams, since we find here that these miRNAs are modulated at d0, as early as the hepatic enzymes involved in lipid metabolism. Furthemore, maternal FFA at gestation correlated directly with miR-370 and inversely with miR-122 in the liver of newborns, thus reinforcing the hypothesis that maternal lipids that cross placental barrier are able to modulate miRNAs expression of offspring.
It have been shown that mice lacking the gene encoding miR-122a are viable, but they develop hepatosteatosis, NASH and fibrosis and present hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokine, including IL-6 and TNFα, thus leading to the development of hepatocellular carcinoma [22, 26, 56]. Besides, Hsu and colleagues (2012) showed that knockout mice for miR-122 in liver present higher expression of several hepatic enzymes involved in TAG synthesis, including Agpat . Additionally, Xu and colleagues showed that inhibition of miR-370 led to downregulation of pro-inflammatory cytokines, suggesting that this miRNA plays a pro-inflammatory role and may be related to hepatic damage . Although the relationship between the levels of miR-122 and miR-370 is still subject to debate [14, 19], we showed that there is an inverse expression of these miRNA in our model at different ages. Thus, considering the impact of lipid metabolism and inflammatory signaling on the development of NAFLD, maternal HFD consumption during the gestational period could be considered an important risk factor for liver diseases in offspring.
Using the cross-fostering model, we were able to further confirm the importance of maternal HFD consumption during gestation to hepatic lipid metabolism and also investigate whether the suckling period would exert the same effect. Surprisingly, miR-122 and miR-370 levels seem to be also modulated by maternal milk, since pups from control dams suckled by HFD-fed dams showed a decrease in miR-122 and increased expression of hepatic miR-370 as well as upregulation of TAG synthesis markers (Agpat and Gpam) and downregulation of a fatty oxidation marker (Acadvl). Regardless of the negative effects of HFD consumption at gestational period seem to be more pronounced, in general, the impairments in lipid homeostasis observed at gestation are also present at the suckling period by itself. Although we did not evaluate milk composition, previous studies demonstrated that HFD consumption and mainly the availability of plasma fatty acid for uptake by the mammary gland alters the fatty acid composition and content, specially medium chain fatty acids secreted in milk of human and rat [42, 58, 59], and may exert effects on the fat accumulation in the neonatal liver.
Besides, offspring from HFD-dam presents altered serum lipid levels  and here, offspring's serum TAG correlated directly with miR-370 and inversely with miR-122 hepatic expression.
Given that we showed that excess maternal lipids at both gestational and lactation periods are independently able to alter hepatic miRNAs in newborn and recently weaned offspring, respectively, that may lead to impaired lipid metabolism, the next step was to investigate whether these modulation would persist into adult life. As shown here and previously, offspring exposed to maternal HFD at gestation and lactation still present, at d82, increased body weight, adiposity, fasting glucose and fat accumulation within the liver. Interestingly, adult offspring from HFD-fed dam still shows decreased miR-122 and increased miR-370 expression in the liver. These findings suggest that epigenetic modifications at pre and recent post-natal life are persistent. Thorn and colleagues (2014) also showed that, in non-human primates, maternal insulin resistance induced by HFD consumption at developmental stages negatively and irreversible affects hepatic immune system and development of de novo lipogenic pathways . For these reasons, authors believes that exposure to excess maternal lipids at critical development periods may be considered the "first hit" for the pathogenesis of liver diseases [60, 61].
The "two hits" or "multiple hits" hypothesis was postulated as an attempt to explain the mechanisms underlying the progression of NAFLD to more aggressive liver diseases, such as NASH and fibrosis. Ectopic accumulation of fat in the liver, the steatosis, was considered the "first hit", and authors suggested that a second hepatic insult, such as oxidative stress or drugs, would be necessary to lead to chronical inflammation and NAFLD progression [62, 63]. Nowadays, it is hypothesized that the obesity and lipid overload in intra-uterine environment could program the foetal liver and trigger the "first hit" and, afterwards, a high-fat diet consumption in post-natal life would be enough to lead to hepatic inflammation and to liver diseases progression .
In this context, we showed here that offspring challenged with maternal lipid overload in pre-natal life and through lactation present a more deleterious response when exposed to a HFD in adult life, by showing an increase in weight gain, adiposity and serum parameters, such as CHOL, TAG, leptin and fasting glucose and, moreover, exacerbated glucose homeostasis disturbances and lipid accumulation in the liver, factors that may lead to inflammatory pathways activation. We showed recently that glucose homeostasis disturbances observed in adult offspring re-challenged to HFD may occur due to an imbalance in insulin signaling in peripheral tissues (such as visceral adipose tissue) and hypothalamus. Additionally a failure in glucose production blockade in the liver was also observed . Therefore, since insulin resistance plays a central role in the metabolic syndrome and the ectopic lipid accumulation in the liver has been considered the hepatic manifestation of this condition [4, 5], our results suggest that offspring from obese dams that are re-exposed to a HFD in adult life could present a metabolic syndrome-like phenotype.
Besides others studies also had shown these increased metabolic disturbances in adult offspring from obese dams exposed to a HFD in adult , to our knowledge, the present study was the first to show that maternal diet leads to persistent modulation of key liver miRNAs that may be involved in the development of insulin resistance and NAFLD. Thus, the attention to maternal diet during gestation and lactation seems to be of great importance to avoid permanent changes during foetal development that could contribute to the development of obesity and its comorbidities in later life.