Altitude, Attitude and Adaptation

Abstract

The fetus has the extraordinary capacity to respond to stress during development, which, in a large part, is mediated by the hypothalamo- pituitary-adrenal (HPA) axis. Hypoxia represents a significant risk to fetal homeostasis and can occur in a wide range of settings including maternal smoking, preeclampsia, preterm labor and high altitude. To study fetal adaptation to chronic, gestational hypoxia, we developed a model of high-altitude, long-term hypoxia (LTH) in pregnant sheep. We discuss the role of LTH on the HPA axis and potential programming of adaptive responses. LTH causes significant activation of the hypothalamic paraventricular nucleus (PVN) and anterior pituitary. In marked contrast, there is an adaptive inhibition in the adrenal, thus balancing the potentially maladaptive centrally mediated responses to LTH. Additionally, we discuss effects of LTH on adipose tissue development. LTH enhances leptin production, which in turn has a regulatory role on the adrenal cortex. Importantly, LTH also has a significant impact on programming of adipose tissue function. Together, our studies show that LTH induces a number of adaptive responses in the ovine fetus. Although they may be beneficial during fetal life, these adaptations could prove to be deleterious in the postnatal period and adulthood.

1. Dr. Ducsay’s Perspective

I first met Dr. Longo at a meeting in Oxford in 1984 and we had a long discussion about fetal physiology. At the time I was doing research on preterm labor at the Oregon Regional Primate Research Center. This was a truly serendipitous meeting, since later that year, Dr. Longo sent out an announcement that the Center was recruiting a reproductive physiologist. Fortunately I got the position and arrived in Loma Linda in December of 1985. Under Dr. Longo’s leadership, I was able to establish an independent research program and receive NIH funding. His mantra was “Do good science and I’ll stay out of the way.” I continued my work on preterm labor, but one day, a simple conversation with Dr. Longo changed my research direction. He mentioned that he had a plan to take pregnant sheep to high altitude and study the effects of long term hypoxia (LTH) on fetal development. Although I hadn’t worked with sheep since graduate school, this seemed like a bad idea. The early work of Mont Liggins, John Challis and others clearly showed that stress (i.e. activation of the fetal hypothalamic-pituitary-adrenal (HPA) axis) would lead to preterm delivery. Well here was Dr. Longo, planning on taking sheep to 13,000 ft, reducing fetal PO₂ by 25 %. This sounded stressful to me; enough so that I said as diplomatically as I could “Dr. Longo, you are going to have preterm lambs strewn all over the side of that mountain. It should be a great model for preterm labor!” Surprisingly, the ewes did not delivery prematurely. This led to our early observations that, in the late gestation LTH fetus, fetal cortisol levels were similar to normoxic controls under basal conditions. However in response to a secondary stressor such as hypotension [1] or umbilical cord occlusion [2] LTH fetuses mounted an enhanced cortisol response. Together, these data suggested that the fetal hypothalamo-pituitary adrenocortical stress axis had adapted to the long term stress of LTH to prevent what I had predicted: an early stress induced maturation of the HPA axis and a premature rise in fetal plasma cortisol, which in this species, drives parturition.

2. Dr. Myers’ Perspective

During my years as a post-doctoral fellow in Dr. Peter Nathanielsz’ laboratory at Cornell, Dr. Longo was one of those individuals at the annual SGI meeting that was larger than life. At the SGI meeting, he always seemed to be surrounded by all the other ‘big names’, a true champion in the perinatal field. It wasn’t until many years later, as a Professor at the University of Oklahoma Health Sciences Center, that I would get to know Dr. Longo personally- thanks to his high altitude sheep! I was talking to Dr. Charles Ducsay from Loma Linda while at an SGI meeting in Washington DC. Charley was telling me about the high altitude fetal sheep preparation that they were working with and my first comment, as someone who had been studying the fetal sheep HPA axis for a decade and a half, was that the hypoxic stress of altitude should cause these sheep to deliver early! Over lunch later that day, he showed me the data from the LTH fetuses and then the response of these fetuses to an acute stressor and I was truly perplexed. We started to discuss the data that my laboratory was presenting at the SGI meeting on maturation of proopiomelanocortin (POMC) processing to ACTH in the fetal anterior pituitary and its regulation by the hypothalamus and how this data might provide a mechanism in the adaptation shown by the LTH fetuses. Charley invited me to give a seminar at Loma Linda, to meet the members of the Center for Perinatal Biology and discuss further potential collaboration on the LTH model. While at Loma Linda for what was to become the first of many, many visits, I was able to finally meet and have a chance to talk science with Dr. Longo. By this time, Charley and I were already discussing my participation on Charley’s project on the resubmission of the NIH Program Project grant that Dr. Longo served as Program director. Dr. Longo fully supported our collaboration and my participation with the Center. Over the years of productive collaboration since those early days, Dr. Longo has made me feel fully at home, often remarking that I’d become part of the furniture of the Center. It’s truly amazing that his insightful concept of high altitude pregnant sheep would have proven so fruitful for the Center and so well recognized for its contributions to fetal and maternal adaptation to long-term hypoxia.

3. The Fetal Hypothalamo-Pituitary-Adrenocortical (HPA) Stress Axis

During pregnancy, the fetus is exposed to a wide range of intrauterine perturbations, indeed stresses, that pose mild to severe threats to fetal health and survival. However, the developing fetus has an amazing ability to respond and adapt to a wide range of potential insults, particularly as its homeostatic mechanisms mature as term gestation approaches. One of the major fetal defense mechanisms is the hypothalamo-pituitary-adrenocortical (HPA) axis. As in adults, the fetal HPA axis, via cortisol, plays a key role in the capacity of the fetus to respond to, and survive these adverse intrauterine events (stressors).

The ovine fetus has been used extensively to study the process of maturation as well as function of the HPA axis. During the final third of gestation, the ovine HPA axis undergoes a slow maturation, during which it gains an increasing capacity to respond to acute stresses. In addition, as the maturation progresses fetal plasma cortisol concentrations undergo an exponential rise culminating at term [3]. Indeed, the preterm glucocorticoid surge is a hallmark of all late gestation mammalian fetuses and essential for critical organ maturation needed to survive the transition to extra uterine life. Maturation of the HPA axis occurs at all levels, with increased expression of the ACTH secretagogues (corticotropin releasing hormone [CRH] and arginine vasopressin [AVP]) in the hypothalamic paraventricular nucleus [4, 5] as well as increased anterior pituitary expression of the ACTH precursor, proopiomelanocortin [6, 7]. This is coupled with its enzymatic processing to ACTH [8], and increased expression of steroidogenic enzymes in the adrenal cortex [9–11], in particular, the rate-limiting enzymes, CYP17 and CYP11A1. The seminal research of the late Dr. Tom McDonald and his colleagues using stereotaxic lesions of the fetal PVN definitively showed that the fetal PVN was essential for the maturation of both the anterior pituitary corticotrope as well as the adrenal cortex [12, 13]. In addition to the late gestation cortisol rise, the increased capacity of the developing fetus to mount a cortisol response to a secondary stressor reflects increased functionality of all aspects of the HPA axis, and is also dependent upon the PVN.

While the preterm rise in fetal plasma cortisol is essential for organ maturation, basal (non-stressed) plasma levels of fetal cortisol are maintained within relatively narrow limits for a given gestational age. Glucocorticoids, like cortisol, are well known for their ability to induce cellular differentiation (hence organ maturation) and therefore, oppose growth. Studies using maternal synthetic glucocorticoid treatment have clearly shown fetal growth restriction in a wide variety of species including rodents [14, 15] sheep [16, 17] and humans [18 ] . Similarly, episodes of acute stress resulting in repeated elevations in fetal cortisol have comparable effects on fetal growth. In sheep and other ruminants, fetal cortisol also plays an essential role of inducing parturition via activation of placental CYP17 expression leading to a decline in progesterone and increase in estradiol [19]. Thus, in these species, the HPA axis provides a mechanism via which a chronically stressed fetus, during the latter portions of pregnancy, can induce both maturation and birth, allowing it to escape the adverse intrauterine environment and increasing chances of survival. In marked contrast, fetal cortisol does not play an active role in parturition in non-human primates. Here, chronic intrauterine stressors lead to elevated basal and stress-induced bouts of fetal cortisol that impair fetal growth and ultimately hinder its chances of survival post birth. This in turn may lead to life long effects, hence programming, as so elegantly predicted by the late Dr. David Barker with his ‘fetal origins’ hypothesis.

Hypoxia is a common fetal perturbation, indeed stressor, which in later stages of gestation can activate the fetal HPA axis. Hypoxia can be considered either acute, ranging in duration from minutes to a few hours, or the so-called chronic hypoxia which is several hours to days in length. In addition, the severity of the hypoxia can vary, from mild to moderate (decreases in PO₂ of ~10–50 %) to severe (50 % or greater). Hypoxic conditions can also be either episodic or sustained. As one might predict, continued exposure to severe hypoxia is often lethal or has lasting deleterious impacts on the fetus and its developing organs, in particular the CNS. Fetuses are commonly, and perhaps routinely, subjected to mild to moderate hypoxia over the course of gestation, in particular in the situations of maternal smoking, preeclampsia, preterm labor, obesity and high altitude. During latter stages of gestation, fetal exposure to moderate sustained or episodic hypoxia in sheep typically results in a robust acute fetal HPA response as well as an advanced maturation of the fetal HPA axis resulting in early delivery of a growth restricted fetus [4, 20]. Indeed, in this model, as one might expect, hypoxia increased expression of CRH and AVP in the PVN, enhanced POMC expression and elevated fetal plasma ACTH leading to a premature maturation of the adrenal cortex and cortisol production [4, 21].

However, what is the impact of sustained fetal exposure to moderate hypoxia initiating prior to period of HPA maturation? In the situations that are commonly associated with moderate long-term (i.e. sustained) hypoxia (LTH) described above, the fetus is exposed to hypoxia prior to maturational phase of homeostatic defense mechanisms. As discussed in the opening paragraphs, Longo and colleagues at Loma Linda developed a model of high altitude (3,820 m, Barcroft Laboratory, White Mountain Research Station, Bishop, CA) induced LTH from approx. 40 days of gestation onward. Fetal PO₂ is maintained at ~18 mmHg (normal is ~23 mmHg) thus creating a moderate hypoxic state for the duration of gestation. Indeed, this is a true state of gestational hypoxia. Since hypoxia, and in particular, moderate hypoxia, is a potent fetal stressor it is remarkable that these fetuses do not exhibit growth restriction or fetal acidosis, and the pregnancies are of normal duration. Our studies have focused on the impact of this LTH on the developing HPA axis and the adaptive mechanisms that have obviously been invoked to permit normal growth and maturation of the fetus in this adverse intrauterine environment.

3.1. HPA Adaptation to the High Life

Our initial studies into the function of the HPA axis in the late gestation LTH fetus were simply quantifying basal ACTH and cortisol concentrations and exploring the HPA response to secondary stressors (severe acute hypotension or umbilical cord occlusion [UCO]). Curiously, since these fetuses had been exposed to LTH since early gestation, resting fetal plasma cortisol concentrations were not different from normoxic control fetuses at the same gestational age of 136–141 days gestation (dG; term is ~145 dG). Thus, the LTH fetus preserves the exponential rise in fetal plasma cortisol during late gestation that is necessary for organ maturation and parturition and helps to explain the lack of growth restriction observed in these fetuses. Also, as predicted by the cortisol concentrations, immune-reactive (IR) ACTH concentrations in the plasma of the LTH fetuses were not different from controls. Thus, the initial indications were that the LTH fetus adapted such that they no longer responded to moderate hypoxia as danger or stress signal.

However, this concept was soon belied as we found that the response of the LTH fetus to secondary stressors, both hypotension and UCO, was resoundingly different compared to normoxic control fetuses. In response to these acute secondary stressors, the plasma cortisol response was significantly elevated vs. control while the plasma IR-ACTH concentrations achieved were similar, indicative of a change in the adrenocortical response to ACTH. However, an alternative explanation existed that centered on ACTH. While plasma IR-ACTH remains relatively constant over the final third of gestation during the period of ACTH-dependent adrenocortical maturation, the “bioactivity” of plasma IR-ACTH increases during this period [22]. Unlike adults where circulating IR-ACTH consists almost entirely of the mature 39 residue ACTH peptide, in fetal sheep, IR-ACTH represents ACTH as well as POMC and partially processed POMC in the form of ACTH-precursors such as the so called 22 kDa proACTH in significant quantities [23, 24]. Both POMC and 22 kDa proACTH have been shown to inhibit ACTH-induced cortisol production in ovine fetal adrenocortical cells [25]. During late gestation there is a progressive PVN-dependent maturation of anterior pituitary corticotrope function including the processing of POMC toward a more adult like profile. Thus, LTH could alter the function of the HPA axis in the fetus at either the level of the hypothalamus and/or anterior pituitary or at the adrenal cortex in its capacity to respond to ACTH.

To explore the initial possibility, we addressed POMC expression and processing in the anterior pituitary as well as the concentrations of ACTH and major ACTH precursors (POMC and 22 kDa proACTH) in LTH fetal plasma, both unstressed and in response to secondary stressors in the LTH fetus. In the anterior pituitary, POMC processing to ACTH was enhanced, as were signs of increased secretion of ACTH (less ACTH stores) [26]. These changes were accompanied with sustained POMC expression. In accordance with our findings at the level of the anterior pituitary, fetal plasma concentrations of ACTH were higher in response to a secondary stress in the LTH fetuses, consistent with the increased cortisol observed during this stimulus. However, unexpectedly, we also observed that basal plasma ACTH was elevated as well in the LTH fetus, despite these fetuses maintaining normal ontogenic concentrations of cortisol in the unstressed state. We also noted that the ratio of ACTH to ACTH precursors was increased, reflective of an enhanced bioactivity of fetal plasma IR-ACTH. Thus, although basal IR-ACTH levels were similar between the LTH and control fetuses, this IR-ACTH was composed of more ACTH and less precursor.

These findings immediately led us to explore our second hypothesis: namely that the adrenal cortex had altered its sensitivity to ACTH accounting for the noted differences in the cortisol response to a secondary stressor. However, we now had to modify our hypothesis. Rather than an increased sensitivity to ACTH, it appears that the adrenocortical cells have adapted to LTH via the capacity to seemingly decrease their sensitivity to circulating ACTH (thus maintaining the normal ontogenic maturation and late gestation rise in fetal plasma cortisol). This seemed to occur while the cells still maintained the capacity to respond to a secondary stress induced increase in ACTH. The latter could be partially answered by the above noted increase in plasma ACTH in the LTH fetus in response to acute stress, but the adaptations accounting for the maintained basal cortisol production in the face of two-fold increased basal plasma ACTH seemed contradictory and somewhat enigmatic. We initially explored expression of key genes that govern various aspects of adrenocortical cortisol synthesis including response to ACTH (the ACTH/melanocortin 2 receptor [MC2R]), delivery of cholesterol to the inner mitochondrial membrane via StAR, where cholesterol is cleaved by CYP11A1 to pregnenolone (first rate limiting step in cortisol synthesis) and subsequent downstream enzymes (CYP17, CYP21, CYP11B1). Despite the elevated basal ACTH, expression of MC2R, CYP11A1 and CYP17 was approximately 50 % less in the LTH fetal adrenal cortex providing one mechanism via which these fetuses escaped the effects of the noted excess plasma ACTH. Although StAR mRNA was not different in the LTH fetal adrenal cortex, the 30 kDa ‘spent’ form of the protein was elevated indicative of an enhanced delivery of cholesterol to CYP11A1, which could help account for the maintained basal production of cortisol.

Based on our findings, the fetal HPA axis responded to LTH as a chronic stressor as might be predicted with a wide range of responses. These included increased CRH and AVP at the level of the hypothalamic PVN (Myers and Ducsay, unpublished observations), enhanced POMC processing to ACTH in the anterior pituitary, as well as enhanced basal ACTH levels [26] and enhanced ACTH in response to an acute secondary stressor [1, 2]. At the same time however, the fetal adrenal exhibited a unique adaptive response to prevent premature maturation of the cortex that would result in early birth of a growth-restricted fetus. Yet, these fetuses retained an enhanced cortisol response to an acute secondary stressor, perhaps reflective of a need for these fetuses ‘living on the edge’ to mount a greater stress response. Deciphering the mechanisms of how the fetal HPA axis adapted to LTH resulting in these responses, in particular the counterintuitive changes noted in the adrenal cortex, was clearly the next step. Identifying the factors mediating these changes could be translated to human pregnancies where a chronically hypoxic, growth restricted fetus was at risk to limit the deleterious actions of sustained moderate hypoxia.

3.2. The Search for the Great Mediator

The adaptive response at the adrenal cortex to LTH clearly involved both intracellular mechanisms as well as extracellular mediators of the hypoxic environment. One such factor is nitric oxide (NO) and we have recently reviewed the role of NO in the regulation of adrenal steroidogenesis in response to LTH [27]. Another novel potential mediator that caught our eyes was the adipose polypeptide hormone, leptin. Leptin had been reported as a physiological suppressor of the HPA axis in adults at both the level of the PVN as well as directly at the adrenal cortex. Both the long, or active splice variant of the leptin receptor (ObRb) as well as short variant (ObRa) of the leptin receptor were found to be expressed in the adult adrenal cortex. Further, leptin had been reported to suppress both ACTH stimulated CYP11A1 and CYP17 expression. In rodent adrenals, leptin also acts to limit corticosterone production via effects on StAR and PBR proteins.

Leptin circulates in fetal sheep, albeit at lower levels compared to adults and in sheep, like humans [28], is primarily produced by the developing adipose tissue. Intracerebral infusion of leptin to late gestation fetal sheep limits both the amplitude and mean levels of ACTH and cortisol pulses. Further studies in fetal sheep by McMillen and colleagues demonstrated that intravenous leptin infusion inhibited the fetal HPA axis, suppressing both the prepartum rise in ACTH and cortisol [29 ] . The effects were reduced as term pregnancy approached. Based on these pharmacological findings of an effect of exogenous leptin on the fetal HPA axis, we initially asked whether leptin was even elevated in the LTH fetus. Indeed, leptin is a hypoxia inducible gene and hypoxia plays a role in adipose expansion and angiogenesis. We reported that fetal plasma leptin as well as perirenal adipose expression of leptin were elevated in the late gestation LTH fetus [30]. In addition, adrenocortical expression of the ObRb was increased in the adrenal cortex of the LTH fetus. Thus, we hypothesized that leptin was poised to be a potential mediator of the adaptive responses we observed in the HPA axis of the LTH fetus.

To explore leptin as a possible mediator of the effects of LTH that we observed on the fetal HPA axis, and in particular on the fetal adrenal cortex, we performed a 4 day infusion of an ovine leptin receptor antagonist to LTH and normoxic control fetal sheep starting at 139 dG [31]. Surprisingly, during the infusion period, the leptin receptor antagonist had no effect on plasma ACTH or cortisol in either the LTH for normoxic control fetuses compared to saline infused controls for either group. Also curious, the leptin receptor antagonist had no effect in the control fetuses on either CYP11A1 or CYP17 expression, even though STAT3 phosphorylation (STAT is a key component of the leptin receptor signaling pathway) was suppressed. However, in the LTH fetal adrenal, CYP11A1 and CYP17 expression was restored to levels similar to control fetuses. This study emphasized a key finding on the role of leptin in both normal function of the late gestation ovine fetal HPA axis: namely that endogenous leptin may not be playing a physiological role in regulating the maturation of the fetal HPA axis since prior studies used pharmacological levels of leptin [29, 32]. Alternatively, leptin may play a role earlier in gestation, in particular when the fetal adrenal cortex undergoes its approximately mid-gestation loss of cortisol production despite maintained ACTH levels. The latter is supported by our observations that younger fetuses at ~120–130 dG have elevated ObRb compared to near term fetuses [33].

A second major discovery from our studies with the leptin antagonist infusion [31] was that while leptin appears to be a mediator of the adaptation observed at the level of the fetal adrenal cortex to LTH, at least in terms of CYP expression, it is not the only mediator since no effect was observed on either ACTH or cortisol levels. Perhaps the most intriguing finding from these series of studies in the LTH fetus was that fetal perirenal adipose, which initiates its differentiation at approximately mid gestation, may be subject to hypoxic modification of function beyond simply increasing production of leptin. Indeed considering the susceptibility of developing adipose and other organs involved in metabolism for programming for later obesity in the offspring by a variety of intra-uterine stressors or maternal conditions, we felt compelled to further explore the impact of LTH on the developing adipose in the ovine fetus.

3.3. Perirenal Fat-Which Color Does LTH Prefer: Brown, Beige or White?

The perirenal fat deposit in sheep and human fetuses is largely considered a brown fat deposit, characterized by high expression of uncoupling protein-1 (UCP1; [34–36]. UCP1 catalyzes adaptive thermogenesis in brown adipose deposits by dramatically increasing the proton conductance of the inner mitochondrial membrane [37]. Expression of UCP1 peaks during the final week of gestation [38, 39], assuring effective thermo-regulation for the newborn extra-uterine environment via non-shivering thermogenesis. In addition to its expression, UCP1 is activated at birth, accompanied by elevated lipolysis and mobilization of available fat stores.

Since we observed that LTH results in the upregulation of perirenal adipose leptin expression leading to elevated fetal plasma leptin [30], we determined if any other brown or white adipose genes were affected by development under these conditions of sustained moderate developmental hypoxia. At 136–139 dG, we noted a significant elevation in expression of UCP1. In addition, we noted increased expression of deiodinase 2 (DIO2; catalyzes the conversion of T₄ to T₃), 11β hydroxysteroid dehydrogenase I (HSD11B1; catalyzes the conversion of cortisone to cortisol), PPARγ, and PGC1α, all hallmarks of the brown fat phenotype [30]. In support of LTH enhancing the functionality of this brown fat deposit in the ovine fetus, we also noted increased expression of the β3 adrenergic receptor (AR) and transcription factors NRF2 and mtTFA, the latter of which regulate expression of genes governing mitochondrial function [40]. However, we have not found evidence of increased mitochondrial numbers in the perirenal fat of the LTH fetus (Myers, DA and Ducsay, CA, unpublished observations). Thus, LTH may simply increase the activity of existing mitochondria in this fat store. Based on these observations, it appears that the LTH environment is preparing these fetuses to more efficiently generate non-shivering thermogenesis in the post-natal environment.

The mechanism(s) governing the increased brown adipose tissue (BAT) phenotype of the perirenal fat deposit in the LTH fetus is presently unknown. Since exogenous glucocorticoids enhance expression of UCP1 and adrenalectomy prevents the late gestation increase in expression of UCP1 in the ovine fetus [38, 41, 42], the noted increase in HSD11B1 may facilitate local metabolism of cortisone to cortisol, representing one mechanism for the enhanced BAT phenotype in the LTH perirenal adipose deposit. Similarly, T₃ is a known mediator of the BAT phenotype [38] and DIO2 expression is elevated in the perirenal adipose of the LTH fetus. Thus, these two tissue specific responses in the perirenal adipose in response to LTH may allow this tissue to increase its BAT phenotype without systemic increases in cortisol or thyroid hormone which would deleteriously impact fetal growth and organ function with potentially life long consequences. An increase in sensitivity to catecholamines via the increased βAR3 expression may play a role in the adaptive response of the perirenal fat to LTH [38, 43].

The fetal perirenal adipose depot, while considered to have a brown fat phenotype due to its high level of UCP1 and other BAT associated genes, is not typical of brown fat since it consists of a mixed population of both multilocular and unilocular fat deposits. Unilocular fat is typical of white fat as opposed to the classic dense multilocular nature of the classic brown fat deposits, and similarly is less vascular. Further, fetal perirenal fat expresses leptin at levels typical for white fat and the expression of leptin is equally distributed in both unilocular adipocytes as well as multilocular adipocytes in this fat store. UCP1 immunostaining appears similarly distributed in fetal perirenal fat. However, other genes that are hallmarks for brown fat are highly expressed in late gestation fetal perirenal fat including the transcriptional co-activators, PRDM16 and PGC1α, and the transcription factor PPARγ, as well as genes such as DIO2 and CIDEA, which are highly expressed in brown fat [44, 45]. In adults, some white adipose tissues can express high levels of UCP1 upon either cold exposure or activation of cAMP signaling pathways e.g., adrenergic stimulation [46, 47]. In addition to UCP1 expression there is an increased multilocular appearance of these fat deposits during the ‘browning’ process. However, while BAT is derived from a myf-5 lineage i.e., muscle cell/BAT precursor, [48–50], these fat deposits are derived from the classic white adipose tissue (WAT) lineage [48–50]. The brown fat like cells in these fat deposits have been referred to as “Beige” or “Brite” cells, and the process via which UCP1 expression is increased has been termed “Beiging” [48–50]. Considering that the perirenal fat rapidly loses its expression of UCP1 and other phenotypic BAT genes (e.g., PGC1α) post birth [45], perirenal fat may represent a true beige deposit and not the classic myf-5 derived BAT.

The increased BAT phenotype of the perirenal fat during late gestation in response to LTH raises an intriguing question considering that increases in BAT in experimental animals is associated with a leaner phenotype while a loss in BAT is associated with obesity and its related metabolic disorders [48, 51, 52]. Thus, our findings have implications for the LTH offspring post-birth in terms of both sensitivity to fat deposition and metabolism if the BAT phenotype of the perirenal fat is maintained. This is especially relevant considering the dramatic increase observed in childhood and adult obesity and metabolic disorders. The ‘fetal origins of adult disease’ hypothesis of Barker [53–55] purports that so-called adverse intrauterine environments can cause permanent epigenetic imprints in the embryo/fetus, predisposing it to susceptibility to a variety of diseases, including obesity and metabolic disorders later in life. Indeed, maternal under nutrition during gestation is linked to obesity and type II diabetes later in life [54, 56]. However, few studies have focused on fetal origins of childhood obesity or of equal importance, early changes in adipose function that can predispose an individual to obesity, metabolic disorders and cardiovascular disease.

An increased risk of obesity and metabolic syndrome has been reported among children born from gestational diabetic or obese mothers [57]. Further, children of obese mothers or mothers with gestational diabetes have a greater neonatal fat mass [58]. A study by Oken et al. [59] reported that the rate for ‘risk for obesity’ was 17.1 % with a 9.7 % ‘obesity’ rate in children of obese mothers, compared to 14.2 % and 6.6 %, respectively, in children of non-obese mothers. Thus, while maternal obesity (gestational and pre-gestation obesity) is strongly linked as a causative factor in childhood obesity [58], it is apparent that not all children born from obese women will develop obesity [58], and a significant population of children from non-obese/diabetic mothers are at high risk or will develop childhood obesity. This emphasizes that other factors contribute to the programming of childhood obesity. Therefore, we propose that fetal hypoxia, via its impact on developing abdominal fat, predisposes the offspring to early fat deposition.

In studies in the LTH fetus, we found that in addition to BAT phenotypic genes, genes governing WAT expansion and function are also upregulated by LTH (e.g., PPARγ, HSD11B1). Thus, we were intrigued as to which phenotypic adaptation, if any, was maintained in the LTH lamb post birth. Surprisingly, by 14 days post birth, the LTH lambs lost the brown fat phenotype significantly compared to the normoxic control lambs [60]. This included a decrease in UCP1 as well as the transcriptional regulators of the brown fat phenotype, PGC1α and PRDM16. However, PPARγ and PPARα expression as well as HSD11B1 expression was similar in the perirenal fat of the LTH and control lambs at 14 days post-birth. Considering that brown and/or beige fat is thought to be protective of adiposity, it appears that the in utero LTH environment may have impacted the perirenal fat in such a manner to favor fat deposition/expansion (PPARγ and HSD11B1) rather than lipid metabolism (UCP1). It will be of considerable interest to see how these lambs respond if provided access to a restricted diet or a diet high in fat and or carbohydrates and to further follow the changes in gene expression in perirenal fat stores as well as visceral and subcutaneous fat.

4. Perspectives/Conclusions

The fetus has the ability to successfully respond to acute stressors. As we have detailed above, it also responds to long-term stressors like high altitude hypoxia. It does so with an “attitude” towards adaptation with a balance between upregulation of the hypothalamic-pituitary axis and a down regulation of adrenal responsiveness at the basal level. One of the mechanisms involved in this adaptation is activation of adipose tissue and enhanced production of leptin. This regulation of basal cortisol is crucial to survival and may also have the added advantage of enhancing the brown fat phenotype in anticipation of birth into a potentially hostile environment. However, in the transition from fetal to neonatal life, there is a shift to an enhanced white fat phenotype that has the potential to result in enhanced adiposity in later life. At present, the mechanism(s) of this unintended consequence of fetal adaptation to altitude remains to be elucidated. However, the results of these studies strengthen the hypothesis that LTH plays a key role in fetal programming with effects long after birth. Dr. Longo always closes his letters and emails with the phrase: “persevere.” It is remarkable that in his LTH sheep that the fetus has done just that!