**4. Discussion**

In this study, we aimed to evaluate the impact of lactation environment on body, cardiovascular and adipose tissue growth in rats with LBW induced by undernutrition during fetal life (MUN offspring). The main findings of the study are summarized in Table 2. During lactation, MUN offspring accelerate body weight gain, while length growth is slower. In males, this growth pattern occurs during the exclusive lactation period, while in females takes place during the last week before weaning, when the rats suckle and eat chow. By weaning, MUN males but not females, exhibit diastolic dysfunction, and heart, aorta, and perivascular white adipocyte hypertrophy; a lower proportion of beige-type adipocytes was found in MUN offspring from both sexes. These sex-dependent alterations may set the basis for the observed hypertension development in MUN males and can predispose to obesity in males and females. We also demonstrated that nursing MUN rats with a controlling mother rescues the hypertrophy alterations, in parallel with a slower weight gain, while nursing control pups, one MUN mother did not have a hypertrophic effect. These data suggest that both MUN rats, in the fetal and perinatal periods, are required to develop phenotypic alterations. In addition, the alterations programmed during the fetal period may be counteracted during lactation by reducing the growth rate. The period of exclusive lactation seems to be more relevant for the observed hypertrophy, even though no major modifications in breastmilk macronutrients were detected between MUN and Control dams, suggesting the role of breastmilk bioactive factors. In summary, the present data provide experimental evidence of the link between accelerated growth during early postnatal life in LBW individuals and higher-risk cardiometabolic diseases and support that modulation of growth during the lactation period can be an effective strategy to counteract alterations induced during fetal life.

**Table 2.** Summary of main findings.


Alterations induced by maternal undernutrition (MUN) are considered if M-on-M was different from C-on-C. Reversal by cross-fostering (CF) in lactation was considered if the alteration induced by MUN (M-on-C) is normalized, being similar to the C-on-C group. If no alterations were found, the effect of lactation was not considered (shown as -). No significant (ns).

Catch-up growth has been proposed to be deleterious for cardiometabolic health in individuals with LBW. We addressed the role of growth during lactation in a rat model of LBW induced by fetal undernutrition, which develops hypertension and cardiac alterations in adult life. In rats, suckling is the only source of nutrition until day 16 of lactation, while from day 17 to weaning there is an increasing content of chow in the stomach of the pups [28]. Therefore, we analyzed growth patterns during both periods of lactation. We found a sexual dimorphism in the growth pattern. In males, accelerated weight gain was observed during the period of exclusive lactation, while in female offspring catch-up growth was observed in the last week of lactation. We also observed that control rats fed on MUN mothers had a slower weight gain both in males and females. To explain these data, we considered differences in milk composition. However, we did not find significant variations in protein or fat content although a tendency toward lower values was observed in MUN dams. It is possible that the milk from MUN dams had lower macronutrient levels at birth, as we have evidenced in plasma [29], but they may normalize along lactation, since the rat returns to an *ad libitum* diet. In other rodent models of programming, milk protein content was found to be unaffected if the dam was fed with low protein [30,31] or with high energy diets [32] during gestation or lactation. We also observed a slower length gain during the last week of the lactation period, during which the rats suckle and also eat chow. We do not think this is related to the lower mineral content of the milk, since minerals seem to be relatively stable [13] and it is possible that there was a prior bone deficiency induced by undernutrition. We also discarded a lower milk yield due to compromised mammary gland development, observed in a rat model of fetal programming induced by uteroplacental insufficiency [33], since it would not explain the quicker weight gain in M-onM offspring. Growth acceleration in MUN males could be explained by hyperphagia which can be programmed [34], is associated with increased neonatal growth rates and visceral adiposity [35], and it has been described in offspring from protein-restricted dams [36]. We do not think dam behavioral alterations, which have been described in SHR and WKY cross-fostering [37], explain our data since all the MUN and Control dams accepted the pups from the other mother successfully. The main influence on growth rate was observed during the period of exclusive lactation; since no major alterations in macronutrients were observed, we suggest the role of milk bioactive components, such as hormones or growth factors. This hypothesis is supported by metabolomic analysis of milk from rats exposed to mild caloric restriction during lactation showing changes in several compounds related to metabolic pathways [38], and the evidence that leptin supplementation during the suckling period reverses some of the metabolic alterations induced by a moderate maternal calorie restriction during gestation [39]. It would be interesting to conduct an in-depth milk metabolomic study on the alterations induced by undernutrition in our rat model.

Rapid growth gain has been related to increased adiposity and obesity development, and therefore, we analyzed possible alterations in body fat. We found that, in parallel with accelerated weight gain, by weaning M-on-M males exhibited hypertrophied perivascular WAT adipocytes compared to controls, an alteration reversed by nursing MUN offspring on a control dam. Similar results were found in a model of obesity programming induced by monosodium glutamate, where cross-fostering also mitigates obesity development [22]. In addition to energy-storing WAT, adipose tissue also comprises thermogenic brown adipose tissue (BAT), and beige adipocytes, an inducible form of thermogenic adipocytes interspersed within WAT [40] recruited postnatally in a process called browning [41]. Browning or "beigeing" has recently gained attention, since beige adipose tissue has a larger energy expenditure capacity, and induced browning in newborn rats decreases adipogenesis in adult life, suggesting a possible way through which the neonatal period can influence obesity development [42]. Therefore, we analyzed the proportion of beige adipocytes within the perivascular WAT in our experimental model, using Mitotracker, a specific dye for mitochondria, since beige adipocytes possess abundant cristae-dense mitochondria [40,43]. MUN rats had a very small proportion of beige-type adipocytes, which may be related to mitochondrial alterations induced by undernutrition, since mitochondrion is a very sensitive organelle and programming has been demonstrated in response to several intrauterine stress factors [44]. A reduction in beige thermogenic tissue could disbalance energetics and facilitate lipid accumulation, leading to the observed increased WAT adipocyte size. It was interesting that beige-type adipocytes increased by fostering MUN rats on a control mother. It has been demonstrated that beige adipose tissue appears spontaneously in WAT during early postnatal development with a peak of expression observed at 21 days [45]. Therefore, the lactation period is an important window during which adipose tissue type may be modulated. It would be interesting to analyze in MUN rats the effect of the lactation environment on adipocyte progenitor markers, such as mesenchymal cell antigen 1 (MSCA1), which has been positively correlated to obesity, adipocyte hypertrophy, and inflammation in children [46]. It is interesting to note that, increased browning occurred in MUN rats exposed to a control lactation environment, while it decreased in control rats exposed to a MUN mother. Therefore, unlike cardiovascular hypertrophy, which seems to require the first hit being during intrauterine life, adipose tissue may be regulated by the lactation environment alone. This is evidenced by our previous study showing that accelerated growth during lactation induced by reducing litter size, without prior programming, also increases WAT deposits [20]. Prolonged retention of thermogenic beige adipocytes maintains high whole-body energy expenditure and protects mice from diet-induced obesity [47]. Therefore, it would be interesting to evaluate if cross-fostered MUN rats retain this characteristic in adult life. Our finding of beige adipocytes within the WAT perivascular tissue is interesting, since, in mice, beige adipocytes are enriched within subcutaneous fat depots, and are rarely detected in visceral depots [43]. Perivascular WAT also plays a role in vascular tone and maintenance of normal structure [43], which is dysregulated by excess

fat, as we have reported in obese mice [48]. A reduced proportion of beige-adipocytes may play a role in vascular dysfunction, an alteration observed in several animal models of fetal programming [49]. These alterations may be due to the pro-oxidant and proinflammatory environment associated with excess fat, favoring the local release of vasoactive factors. Thus, alterations in the proportion of beige-to-WAT perivascular adipose tissue may contribute to the association between obesity and cardiovascular disease [50], an aspect that deserves further attention. The cardioprotective effect of beige adipose tissue is suggested by the fact that individuals with detectable thermogenic adipose tissues have lower odds of hypertension and coronary heart disease [51].

Accelerated body growth gain in MUN males was paralleled by a hypertrophic response in the cardiovascular system observed in the aorta and the heart. The characteristics of the structural alteration in the aorta are compatible with an outward hypertrophic remodeling, with an increase in wall mass due to expansion of the external diameter. Females show a better adaptation, although the data also evidence a tendency towards a larger growth, as we have previously described in resistance arteries [52]. Undernutrition induced marked heart alterations by weaning, in addition to ventricular hypertrophy, a reduced diastolic function was also observed, suggesting that cardiac tissue has a high susceptibility to programming, also observed in guinea pigs exposed to undernutrition, which show a permanent deficiency in cardiomyocyte number [53]. We did not explore molecular markers of hypertrophy, such as BNP; we have evidence that this factor is not elevated in MUN offspring by weaning, but it is increased in aging in MUN males along with hypertension development and further cardiac hypertrophy [18]. The role of sex in heart alterations induced by undernutrition may be related to the lower efficiency of MUN male placenta, associated with poor vascularization [29], which may be particularly detrimental for the heart due to the unique feature of cardiomyocytes as non-dividing cells [54]. Cardiovascular hypertrophy in MUN males was rescued by crossfostering, reinforcing the role of lactation in reprogramming cardiovascular disease. The importance of this period for future cardiometabolic health is supported by data in the genetic model of essential hypertension, the SHR rat, which reduces blood pressure levels in adult life if cross-fostered to a control dam [6]. It is worth mentioning that this model of essential hypertension also exhibits sexual dimorphism and we have found several common features with the MUN model [55].

#### **5. Conclusions**

The lactation period acts as a second hit consolidating programming initiated by fetal undernutrition in a sex-dependent manner, inducing cardiovascular and adipocyte hypertrophy.

The alterations programmed during fetal life may be counteracted during the perinatal period avoiding accelerated growth and thus, lactation and can be a window of intervention to reverse fetal programming.

The exclusive lactation period seems to play a key role in organ hypertrophy, despite no differences in breastmilk macronutrients, suggesting the role of bioactive factors, which deserves in-depth analysis.

**Author Contributions:** Conceptualization, S.M.A., P.R.-R., I.M.-C. and D.R.-C.; methodology, P.R.-R., I.M.-C., S.P., B.Q.-V., A.G.-R. and S.C.; software, I.M.-C. and D.R.-C.; validation, P.R.-R., I.M.-C., D.R.-C., S.P., B.Q.-V., A.G.-R. and S.M.A.; formal analysis, D.R.-C.; investigation, P.R.-R., I.M.-C., S.P., B.Q.-V., A.G.-R. and S.C.; resources, S.M.A.; data curation, P.R.-R., I.M.-C., S.R. and S.P.; writing original draft preparation, S.M.A. and D.R.-C.; writing—review and editing, P.R.-R., I.M.-C., S.P., B.Q.-V., S.R., A.G.-R., S.C., D.R.-C. and S.M.A.; visualization, P.R.-R., I.M.-C., D.R.-C. and S.M.A.; supervision, S.M.A.; funding acquisition, S.M.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministerio de Ciencia, Innovación y Universidades (Spain; grant number RTI2018-097504-B-I00), co-financed with FEDER funds.

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Review Board of Universidad Autónoma de Madrid and Comunidad Autónoma de Madrid (Spain, protocol code PROEX 004-19 and approved on 7 May 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The availability of the data is restricted to investigators based in academic institutions.

**Conflicts of Interest:** The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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