Maternal deprivation

Weaning in laboratory animals occurs at various time-points depending upon the species/strain and ethical regulations, but frequently in rodents, “standard weaning” will occur on postnatal day (PND) 21.^{Reference Curley, Jordan, Swaney, Izraelit, Kammel and Champagne59,Reference Sengupta60} At this age, the offspring show a degree of independence and spend more time eating solid food than suckling.^{Reference Sengupta60} This rodent weaning procedure is performed by separating the dam from her litter. Therefore, the separation of a mother and her litter before PND21 is a model of early weaning.

Early weaning by maternal deprivation (MD) has an impact on the metabolism and behavior of offspring throughout life.^{Reference Kikusui, Nakamura, Kakuma and Mori61–Reference Nakagaki, Mafra and de Carvalho65} This model involves maternal milk restriction and maternal care restriction, which promotes perinatal stress.^{Reference Ghizoni, Figueiredo, Moisan, Ogias, Osaki and Gama66} Therefore, the offspring outcomes could be adaptive responses to nutritional changes as well as emotional stress.

The changes in the hippocampus–hypothalamus–pituitary–adrenal (HHPA) axis of offspring after MD highlight the role of perinatal stress in this model. Thus, neonatal changes in corticosterone levels or signaling are involved in many metabolic programming models, and this hormone is a candidate imprinting factor.^{Reference Macrì67,Reference Weaver, Cervoni and Champagne68} In rodent models, early weaning at PND14–15 increased serum corticosterone 2 d after maternal separation^{Reference Ghizoni, Figueiredo, Moisan, Ogias, Osaki and Gama66} and promoted increased activity and decreased resting behavior over the period from PND15 to 21, revealing stress-induced behavior in both sexes.^{Reference Kikusui, Isaka and Mori69} In adulthood, early-weaned offspring maintained increased serum corticosterone levels in basal or stressful conditions,^{Reference Kikusui, Nakamura, Kakuma and Mori61,Reference Kikusui, Takeuchi and Mori70} increased anxiety,^{Reference Kikusui, Isaka and Mori69} and aggressiveness were exhibited by both sexes.^{Reference Kikusui, Nakamura, Kakuma and Mori61,Reference Kikusui, Takeuchi and Mori70} In addition, adult female offspring showed decreased maternal behavior with offspring.^{Reference Kikusui, Isaka and Mori69}

In addition to the behavioral changes, the metabolic outcomes in rat offspring were initially exhibited as a reduced body weight, which remained present^{Reference Crispel, Katz, Ben-Yosef and Hochberg62} or became normal in adulthood (150 d old).^{Reference dos Santos Oliveira, de Lima, da Silva, da Silva, de Souza and Manhães-de-Castro63,Reference Boueri, Pessanha and Da Costa71} Interestingly, early-weaned animals showed increased glucose tolerance and insulin sensitivity.^{Reference Crispel, Katz, Ben-Yosef and Hochberg62} However, these animals exhibited changes in the behavioral satiety test compared to late-weaned animals (PND31), suggesting a tendency toward delayed satiety behavior.^{Reference dos Santos Oliveira, de Lima, da Silva, da Silva, de Souza and Manhães-de-Castro63} In addition, in adult life, the early-weaned rats showed increased hepatic lipogenesis and hepatic cholesterol without changes in glucose tolerance and plasma cholesterol concentrations.^{Reference Back and Angel64} Interestingly, the hepatic alterations in response to early weaning could be a result of profound changes in the expression of several liver metabolic enzymes, starting with those involved in hepatic metabolic function.^{Reference Nakagaki, Mafra and de Carvalho65} Early weaning decreases hepatic immune function and accelerates the shift in metabolic functioning during neonatal development, which may affect liver function throughout life.

Interestingly, small episodes of maternal separation (4–8 h) before MD at PND17 in the maternal separation and early weaning (MSEW) model show how early-life neglect is reflected in the behavioral changes observed in neglected children, including hyperactivity, anxiety, and attention deficits.^{Reference Carlyle, Duque and Kitchen72} The MSEW female mice exposed to a high-fat diet (HFD) showed increased body weight, adiposity, and fasting glucose levels.^{Reference Murphy, Herald, Leachman, Villasante Tezanos, Cohn and Loria73} Moreover, these animals exhibited hyperinsulinemia, hyperleptinemia, and hypertension.^{Reference Murphy, Herald, Leachman, Villasante Tezanos, Cohn and Loria73} Therefore, MD increased the susceptibility to obesity and metabolic disorders in offspring in response to HFD, especially in female offspring.

Early-life stress is an important factor involved in metabolic programming. Therefore, not only is breast milk deprivation involved in offspring outcomes but also emotional stress resulting from MD. Punctual maternal separation for 24 h in PND10 induces HPA axis hyperactivity, exacerbating the response to stress in adult offspring.^{Reference Clarke, Cai, Saleh, Buller and Spencer74} Short maternal separation during the lactation period (10 min daily of maternal separation plus stress) also activated the HPA axis at weaning and in adult life^{Reference Loizzo, Loizzo and Galietta75} and promoted overweight, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and hyperleptinemia in early-weaned offspring in adulthood.^{Reference Loizzo, Loizzo and Galietta75}

Early weaning by MD is an interesting model of early weaning that mimics the real condition of mothers who abandon their child. The repeated lack of contact between mother and litter in rodent models reflects early-life child neglect, which is currently a social health challenge. Therefore, as the restriction of maternal care alone affects offspring development, rodent models of early weaning with no maternal separation could attenuate emotional stress by isolating the impact of breastfeeding restriction on offspring outcomes. For some years, our laboratory has been dedicated to studying the effects on programming of early weaning without MD to better understand the mechanisms underlying the increased risk for the development of obesity and its comorbidities. Fig. 2 depicts some results already published in the pharmacological and nonpharmacological models of early weaning, which are detailed below.

Fig. 2. Similarities and differences in metabolic outcomes between pharmacological early weaning (PEW) and nonpharmacological early weaning (NPEW) adult male offspring. The early weaning models showed some similarities; however, they could promote different offspring outcomes throughout life. The differences between PEW and NPEW adult male offspring are highlighted in red. Legend: brown adipose tissue (BAT), sympathetic nervous system (SNS), uncoupled protein 1 (UCP1).

Pharmacological early weaning

The inhibition of breast milk production using a pharmacological approach is seen as another experimental model of early weaning. Some drugs, such as bromocriptine, a dopamine-2-receptor agonist, are known for their rapid inhibitory effect on prolactin production at the pituitary level,^{Reference Ben-Jonathan and Hnasko76} promoting the reduction of maternal milk biosynthesis. In a rat model, maternal bromocriptine administration for the last 3 d of lactation showed potent effects in inhibiting prolactin, reducing milk production and, consequently, directly impacting the pup body weight at PND21.^{Reference Bonomo, Lisboa, Passos, Pazos-Moura, Reis and De Moura77} It is important to note that the pups received less milk but still received chow pellets directly in the cage. Therefore, the reduction of breast milk production by bromocriptine administration in late lactation is considered a pharmacological early weaning model (PEW), which plays a potent role in offspring metabolic programming.

Similar to MD, the PEW model is also a model of early-life stress, highlighted by the increased serum corticosterone level at PND21^{Reference Fraga, Moura and Silva78} and in adult life.^{Reference de Moura, Bonomo and Nogueira-Neto79} In addition, these animals exhibited higher catecholamine content in the adrenal gland in adulthood.^{Reference de Moura, Bonomo and Nogueira-Neto79} These hormonal changes could be involved in behavioral changes during adult life, as indicated by intense anxiety-like behavior and reduced locomotor activity.^{Reference Fraga, Moura and Silva78}

Maternal hypoprolactinemia could alter milk leptin transfer to offspring,^{Reference Bonomo, Lisboa, Passos, Pazos-Moura, Reis and De Moura77} and this effect could modify hypothalamic circuits involved in satiety and energy expenditure^{Reference Vickers80} during the development of hypothalamic and hippocampal circuits.^{Reference Coupé, Dutriez-Casteloot and Breton81} At PND21, PEW offspring had higher plasma leptin.³³ Despite normal levels at PND22, the PEW offspring showed increased leptin levels at PND30^{Reference Carvalho, De Oliveira and Peixoto-Silva82} that persisted until adulthood (PND180).^{Reference Bonomo, Lisboa, Pereira, Cottini, Passos and Gaspar de Moura83} Interestingly, at PND22, the hypothalamus of PEW offspring seemed to be more sensitive to leptin, since leptin receptor (OBR) and signal transducer and activator of transcription 3 (STAT3) protein expression were increased.^{Reference Carvalho, De Oliveira and Peixoto-Silva82} These leptin changes could be implicated in metabolic disorders promoted by developmental programming by changing energy intake and expenditure control.^{Reference Vickers80} Indeed, the early weaning model promotes many metabolic disorders, such as overweight, increased visceral adiposity,^{Reference Bonomo, Lisboa, Pereira, Cottini, Passos and Gaspar de Moura83} hyperglycemia, hypoadiponectinemia, insulin resistance, and dyslipidemia.^{Reference de Moura, Bonomo and Nogueira-Neto79,Reference Peixoto-Silva, Conceicao and Carvalho84} Our group exhaustively investigated the mechanisms involved in the susceptibility to obesity. Although the adult animals showed resistance to the anorectic effect of leptin under challenge, they did not show food intake changes in basal conditions,^{Reference Bonomo, Lisboa, Pereira, Cottini, Passos and Gaspar de Moura83} suggesting that hyperphagia is not the mechanism responsible for the elevation in body weight. Additionally, adult PEW offspring did not show alterations in canonical leptin signaling in the hypothalamus,^{Reference de Moura, Bonomo and Nogueira-Neto79} which does not exclude the participation of other STAT3-independent pathways, such as the phosphoinositide 3-kinase (PI3K) pathway. However, these animals exhibited markers of susceptibility to the disturbance of satiety mechanisms, such as the increased expression of neuropeptide Y (NPY), an orexigenic peptide, in the arcuate nucleus (ARC) and paraventricular nucleus (PVN) of the hypothalamus,^{Reference Younes-Rapozo, Moura, Manhães, Peixoto-Silva, De Oliveira and Lisboa85} and the presence of astrogliosis, which might indicate hypothalamic inflammation.^{Reference Younes-Rapozo, Moura, Manhães, Peixoto-Silva, De Oliveira and Lisboa85} These changes are in accordance with leptin resistance in this model. Therefore, in the PEW model, it is possible that the mechanisms of satiety are borderline effective in promoting food intake changes. Perhaps, after a challenge with palatable foods, for example, these animals may develop hyperphagia.

Additionally, in the PEW model, disturbance in the energy expenditure mechanisms might be involved in the susceptibility to obesity. In adult life, these offspring showed reduced thyroid hormone, which is an important regulator of energy expenditure.^{Reference Ortiga-Carvalho, Chiamolera, Pazos-Moura and Wondisford86} Hypothyroidism in this model was characterized by a reduction in serum triiodothyronine (T3), thyroxine (T4), and thyrotropin (TSH, the major regulator of thyroid hormone synthesis), indicating the central disturbance of the thyroid.^{Reference Bonomo, Lisboa, Passos, Alves, Reis and de Moura87} Hypothyroidism could impair the thermogenic activity of brown adipose tissue (BAT) along with reducing sympathetic nervous system activity in the BAT and reducing adrenergic receptor content (β3-AR) in the PEW offspring.^{Reference Peixoto, Pietrobon and Bertasso88} Despite these changes, the BAT of PEW offspring did not show a change in uncoupled protein 1 (UCP1) expression but showed reduced peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) expression.^{Reference Peixoto, Pietrobon and Bertasso88} Taken together, these changes confirm the presence of hypometabolism in PEW offspring, indicating their susceptibility to obesity. Interestingly, the restoration of serum thyroid hormones and corticosterone levels by chronic low-intensity exercise during life attenuated obesity and its related disorders, such as dyslipidemia and hyperglycemia, in adult male PEW offspring.^{Reference Boaventura, Casimiro-Lopes, Pazos-Moura, Oliveira, Lisboa and Moura89} Although they showed beneficial responses to chronic exercise, PEW offspring showed increased serum lactate, suggesting an impaired capacity of cell membrane adaptation to chronic exercise load.^{Reference Boaventura, Casimiro-Lopes, Pazos-Moura, Oliveira, Lisboa and Moura89} These animals did not show changes in physical performance in response to acute exercise.^{Reference Casimiro-Lopes, Lisboa, Koury, Boaventura, Passos and Moura90}

Liver injury is a common finding in metabolic programming models,^{Reference Miranda, De Almeida and DaRocha91,Reference Franco, Lisboa and Lima92} and due to the hepatic control of carbohydrates and lipid metabolism, this injury is normally involved in associated macronutrient disorders. Interestingly, although the adult offspring exhibited dyslipidemia and glucose intolerance, the liver morphology was conserved in adult PEW offspring, despite the higher hepatic triglyceride levels. The liver in PEW offspring also showed a better redox state than that in control offspring, reaffirming the absence of liver injury at this age.^{Reference Peixoto-Silva, Conceicao and Carvalho84} This intriguing observation might be a result of the beneficial direct hepatic effects of bromocriptine, as observed in another experimental model.^{Reference Davis, Pei and Trush93,Reference Popovic, Janicijevic-Hudomal, Kaurinovic, Rasic and Trivic94}

We also described the gradual dysfunction of the renal physiology of adult offspring of adult PEW offspring, which occurred without changes in blood pressure^{Reference Passos, Passos and Oliveira95} or bone metabolism.^{Reference De Albuquerque Maia, Lisboa, De Oliveira, Da Silva Lima, Da Costa and De Moura96,Reference de Albuquerque Maia, Lisboa and de Oliveira97} Although the adult animals showed a reduction in total bone mineral density and mineral content at PND21,^{Reference De Albuquerque Maia, Lisboa, De Oliveira, Da Silva Lima, Da Costa and De Moura96} they also showed higher total bone mineral density and mineral content along with increased serum 25-hydroxyvitamin D,^{Reference de Albuquerque Maia, Lisboa and de Oliveira97} which could be related to increased serum leptin levels that have been shown to play a protective role and to exhibit a positive correlation with the metabolism of bone.^{Reference Steppan, Crawford, Chidsey-Frink, Ke and Swick98}

The main clinical indication for bromocriptine is as a therapeutic agent for the treatment of prolactin-secreting tumors.^{Reference Schlechte99} Exclusive breastfeeding can be successfully established in babies whose mothers are receiving bromocriptine for the treatment of hyperprolactinemia during pregnancy and lactation,^{Reference Verma, Shah and Faridi100} but it is important to consider its adverse effects. Therefore, the PEW model is able to mimic this early-life exposure to bromocriptine. The offspring outcome from the PEW model could be a result of direct bromocriptine action in offspring and may not only be due to milk restriction. However, offspring that received bromocriptine from PND11 to PND20 of lactation showed a different outcome compared to that of PEW offspring (from mother who received bromocriptine), which showed hyperphagia and hyperthyroidism in adult life.^{Reference Carvalho, Lisboa and de Oliveira101} Therefore, PEW offspring outcomes throughout life are a result of milk restriction, but we cannot discard the influence of direct bromocriptine action. Therefore, an early weaning model without pharmacological intervention and the maintenance of maternal care could provide clear information about maternal milk restriction at early life by mimicking the common human condition of early weaning.

Nonpharmacological early weaning

The nonpharmacological early weaning (NPEW) model is closer to common human early weaning conditions and is without confounding factors, such as high stress or drug side effects. The NPEW model is performed in the last 3 d of lactation by introducing a physical barrier using a breast bandage to interrupt nipple suction.^{Reference Lima, de Moura and Passos102} In contrast to the other early weaning models, this model did not promote acute stress in offspring, since the basal serum corticosterone level at PND21 and in adults was unchanged,^{Reference Lima, Moura and Franco103} reinforcing the isolated impact of maternal milk restriction during late lactation on offspring outcome. Indeed, we did not observe behavioral changes in adulthood.^{Reference Fraga, de Moura and da Silva Lima104}

Although they were not exposed to early-life stress, the NPEW offspring showed decreased body weight and length, adiposity, and serum glucose and serum insulin levels at PND21, which are changes related to milk deprivation.^{Reference Lima, de Moura and Passos102} At this age, the animals showed hypoleptinemia, hypothyroidism (low T3 syndrome), and unaltered adrenal catecholamine content.^{Reference Lima, Moura and Franco103} The T3 reduction could be a strategy to reduce metabolism and, therefore, energy expenditure during the period of energy restriction. In adult life, the NPEW offspring initially exhibited a normal body weight at PND120.^{Reference Nobre, Lisboa and Lima105} However, the body weight was increased throughout life, and the animals were overweight and showed increased total and visceral adiposity at PND180.^{Reference Lima, de Moura and Passos102} In addition, at PND180, the NPEW offspring exhibited metabolic disorders such as hyperglycemia, insulin resistance, hypertriglyceridemia,^{Reference Lima, de Moura and Passos102} and hypertension.^{Reference Franco, Lisboa and Lima92} The serum thyroid hormones and corticosterone levels were normal in adult NPEW offspring, but they showed increased adrenal catecholamine content and increased adrenergic β-3 receptor (β3-AR) expression in adipose tissue, suggesting the presence of reduced levels of catecholamines in serum and reduced tissue effects.^{Reference Lima, Moura and Franco103}

This model has an important impact on hypothalamic circuits involved in the control of satiety. The adult PEW offspring showed increased serum leptin and hypothalamic leptin resistance,^{Reference Lima, de Moura and Passos102} as indicated by increased NPY and decreased cocaine- and amphetamine-regulated transcript (CART) expression, particularly in the PVN.^{Reference Younes-Rapozo, De Moura and Da Silva Lima106,Reference Lima, Franco and Peixoto-Silva107} In addition, these animals showed hypothalamic inflammation, which was highlighted by the increased tumor necrosis factor alpha (TNF-α) expression in the ARC nucleus.^{Reference Lima, De Oliveira, Da Silva, Maia, De Moura and Lisboa108} These central changes explain the basal hyperphagic behavior^{Reference Lima, de Moura and Passos102} and the disturbance in the anorexigenic response after leptin challenge.^{Reference Lima, De Oliveira, Da Silva, Maia, De Moura and Lisboa108} Interestingly, milk restriction altered the hypothalamic circuitry not only in the long term but also in the short term, as observed by increased hypothalamic NPY expression after PND21.^{Reference Younes-Rapozo, De Moura and Da Silva Lima106}

Another satiety mechanism disrupted in NPEW offspring in adulthood is the expected increase in serum glucagon-like peptide 1 (GLP-1) levels after a meal, which stimulates anorexigenic neurons.^{Reference Quitete, Nobre, Peixoto-Silva, de Moura, Lisboa and De Oliveira109} This change might contribute to the hyperphagic phenotype. Early changes in this satiety mechanism were also observed, and it seems to be involved in the adaptation to malnutrition early in life. At PND21, the increased GLP-1 activity in the hypothalamus and adipose tissue plays an adaptive role by imprinting a thrifty phenotype.^{Reference Quitete, Nobre, Peixoto-Silva, de Moura, Lisboa and De Oliveira109} Interestingly, these changes and other metabolic features were reversed by chronic calcium supplementation in adulthood without an impact on hyperphagia,^{Reference Nobre, Lisboa and Lima105,Reference Quitete, Nobre, Peixoto-Silva, de Moura, Lisboa and De Oliveira109} revealing the contribution of other mechanisms in this programming model, such as hypothalamic leptin resistance.^{Reference Lima, de Moura and Passos102,Reference Lima, De Oliveira, Da Silva, Maia, De Moura and Lisboa108}

In addition to hyperphagic behavior, changes in energy expenditure might contribute to obesity in NPEW adult offspring. Although serum thyroid hormone levels were normal in adulthood, the thermogenic activity of BAT might be disturbed in NPEW offspring. We observed reduced sympathetic nervous system activity in BAT, followed by reduced UCP1 and PGC1-α expression.^{Reference Peixoto, Pietrobon and Bertasso88} BAT function also seemed to be compromised by a reduction in the pAMPK/AMPK ratio^{Reference Peixoto, Pietrobon and Bertasso88}; taken together, the BAT changes could promote hypometabolism, contributing to the obesity phenotype.

The NPEW model also exhibited dysfunction in organs involved in metabolic disorders. In addition, white adipose tissue is affected by early weaning. In adult life, the white adipose tissue of NPEW offspring exhibited hypertrophic adipocytes, the increased expression of adipogenesis markers, such as CCAAT/enhancer-binding protein beta (C/EBPB) and peroxisome proliferator-activated receptor gamma (PPAR-γ), and the increased expression of inflammatory markers, such as interleukin 6 (IL-6), TNF-α and monocyte chemotactic protein 1 (MCP1).^{Reference Lima, De Oliveira, Da Silva, Maia, De Moura and Lisboa108,Reference Nobre, Lisboa and Peixoto-Silva110} We also observed markers of reduced vitamin D signaling^{Reference Nobre, Lisboa and Peixoto-Silva110} and increased glucocorticoid signaling^{Reference Miranda, Pietrobon and Bertasso111} in the visceral compartment; however, we also observed normal serum corticosterone levels,^{Reference Lima, Moura and Franco103} which could contribute to increased adipogenesis and lipogenesis in this tissue.^{Reference Nobre, Lisboa and Peixoto-Silva110,Reference Miranda, Pietrobon and Bertasso111} Adipose tissue dysfunction could be involved in the differential expression of adipocytokines via decreased adiponectin and increased leptin content^{Reference Lima, Moura and Franco103} and thereby impact serum hormone levels.^{Reference Lima, de Moura and Passos102} The white adipose tissue of NPEW offspring also showed increased expression of β3-AR, and its upregulation could be due to decreased serum catecholamines, which could be involved in decreased lipolysis in these animals.^{Reference Lima, Moura and Franco103}

Interestingly, although they showed reduced bone mineral density at PND21,^{Reference De Albuquerque Maia, Lisboa, De Oliveira, Da Silva Lima, Da Costa and De Moura96} the adult NPEW offspring showed beneficial changes in bone structure and metabolism. They exhibited increased total bone mineral density and mineral content, including improved bone microarchitecture, and these changes improved the bone biomechanical properties.^{Reference de Albuquerque Maia, Lisboa and de Oliveira97}

In adult life, the liver of NPEW offspring showed increased levels of markers of protein oxidation and lipid peroxidation and decreased antioxidant activity of glutathione peroxidase (GPx)^{Reference Franco, Lisboa and Lima92} and superoxide dismutase (SOD),^{Reference de Oliveira, Lima, Conceição, Peixoto-Silva, Moura and Lisboa112} revealing an imbalance in the redox state. Indeed, the liver morphology revealed steatosis, which was reinforced by increased levels of hepatic triglycerides.^{Reference Franco, Lisboa and Lima92} Liver dysfunction is likely to be involved in hypertriglyceridemia and hyperglycemia in NPEW offspring in adulthood.^{Reference Lima, de Moura and Passos102} These hepatic alterations (liver oxidative stress and microsteatosis) are in contrast to the observed PEW offspring phenotype, suggesting the protective effects of early bromocriptine exposure on the metabolic programming of liver function. Indeed, the NPEW offspring treated with bromocriptine during the last 3 d of lactation were protected from steatosis and glucose intolerance.^{Reference Peixoto-Silva, Moura and Carvalho113} The beneficial effects of bromocriptine on the liver were described before in adult animals.^{Reference Davis, Pei and Trush93,Reference Popovic, Janicijevic-Hudomal, Kaurinovic, Rasic and Trivic94} In addition, early bromocriptine exposure attenuated hyperphagia and increased adiposity and hyperleptinemia in NPEW offspring.^{Reference Peixoto-Silva, Moura and Carvalho113} These observations highlight the deleterious impact of isolated breast milk restriction on liver metabolism, reinforcing the effects of early weaning on the hepatic gene expression profile and function throughout life.^{Reference Nakagaki, Mafra and de Carvalho65}

Although these models mimic the human conditions of early weaning by promoting early life breast milk restriction and the thrifty phenotype throughout life, the hepatic outcome is not the only difference between adult PEW and NPEW offspring. In Fig. 2, we show the similarities and differences between these rodent early weaning models in terms of the function of metabolic tissues and outcomes. Despite similar outcomes for overweight and adiposity, the outcomes for metabolic disorders and hormonal changes were different in their effects and intensity.

Interestingly, we have shown that these metabolic disorders could be attenuated by chronic nutritional interventions during the adult life of NPEW offspring. Hepatic injury was reversed by resveratrol supplementation in adulthood^{Reference Franco, Lisboa and Lima92} and by treatment with Ilex paraguariensis (yerba mate),^{Reference de Oliveira, Lima, Conceição, Peixoto-Silva, Moura and Lisboa112} while adipose tissue dysfunction was prevented by calcium supplementation.^{Reference Nobre, Lisboa and Peixoto-Silva110}

Currently, the major reason for a short breastfeeding duration is the early return to work,^{Reference Ogbuanu, Glover, Probst, Liu and Hussey114} which makes an important contribution to the increased prevalence of early weaning^{Reference Victora, Bahl and Barros50} due the increased number of women in the workforce and plays an important financial role in the family.^{Reference Chai, Nandi and Heymann49} In this situation, breast milk restriction is the major imprinting factor involved, and its isolated impact on offspring metabolism is well represented in the NPEW model, highlighting the importance of exploring the observed mechanisms involved in metabolic programming.

Sex-related differences

As previously mentioned, the majority of data available concerning early weaning have been reported from male animals. However, recently, female offspring in early weaning models were investigated, and, in general, the findings were different, suggesting a sex dimorphism for some outcomes.

Tables 1 and 2 depict the findings in early-weaned female offspring compared to the findings in males. Female offspring in both pharmacological and NPEW models exhibited higher adiposity and hyperphagia, despite having normal body weight. A sex-dependent mechanism seems to be involved in this phenotype because the females showed differences in hormonal profiles,^{Reference Pietrobon, Bertasso and Silva115} BAT thermogenic capacity,^{Reference Peixoto, Pietrobon and Bertasso88} fat deposit distribution, and glucocorticoid status.^{Reference Miranda, Pietrobon and Bertasso111}

Table 1. Different metabolic parameters in offspring of both sexes in a pharmacological early weaning (PEW) model

These data were described by Miranda et al., 2019; Pietrobon et al., 2019; and Peixoto et al., 2019. Legend: White adipose tissue (WAT); 11β-hydroxysteroid dehydrogenase (11β-HSD1); glucocorticoid receptor (GRα); peroxisome proliferator-activated receptor gamma (PPARγ); fatty acid synthase (FAS); brown adipose tissue (BAT); β3-adrenergic receptor (β3-AR); thyroid hormone receptor β1 (TRβ1).

Table 2. Different metabolic parameters of offspring of both sexes in a nonpharmacological early weaning (NPEW) model

These data were described by Miranda et al., 2019; Pietrobon et al., 2019; and Peixoto et al., 2019. Legend: White adipose tissue (WAT); 11β-hydroxysteroid dehydrogenase (11β-HSD1); glucocorticoid receptor alpha (GRα); brown adipose tissue (BAT); sympathetic nervous system (SNS); β3-adrenergic receptor (β3-AR); thyroid hormone receptor β1 (TRβ1).