Characterisation of the Maternal Response to Chronic Phase Shifts during Gestation in the Rat: Implications for Fetal Metabolic Programming

It has become increasingly clear that early environmental influences can have long lasting impact on the normal development and physiology of the fetus. Factors such as poor maternal nutrition, prenatal stress and exposure to medicinal and social drugs can all have negative health consequences for the offspring that persist into adulthood [1]–[3]. This led us to consider whether exposure to abnormal lighting conditions during pregnancy, as occurs during maternal shift work, can also have life-long impacts on the developing fetus.

Due to the wide variations in the types and durations of working schedules conducted in the community, it can be difficult to experimentally expose either human subjects or animal models to ‘shift work’. Nevertheless, shift work is characterised by forced disruptions in the timing of activity, sleep and light exposure, leading to disordered endocrine, metabolic and behavioural rhythms. While it is known from epidemiological and experimental studies that these changes can increase the risk to the individual of developing a myriad of chronic health disorders, it also raises the important question of whether exposure to shift work during pregnancy can also affect the developing fetus.

To address this question, we recently exposed pregnant rats to a chronic phase shift (CPS) protocol whereby the photoperiod was reversed twice every week throughout gestation and for 1 week after birth. We demonstrated that in utero and early postnatal exposure to this protocol negatively affected metabolic homeostasis, with offspring displaying age and gender dependent hyperleptinaemia, hyperinsulinaemia, poor glucose tolerance, insulin resistance and increased adiposity [4]. These changes occurred despite there being no effect of the protocol on birth weight or early postnatal growth. Manipulating the photoperiod in this way is likely to disrupt behavioural, endocrine and metabolite rhythms, as well as deregulate the expression of clock genes. How these disruptions translate into altered metabolic programming in the offspring, however, is unknown.

In this study we investigated the impact of chronic phase shifts on maternal and fetal hormonal, metabolic and circadian rhythms. Pregnant rat dams were exposed to either a control photoperiod or chronic phase shifts of the photoperiod, and maternal weight gain, food consumption, the circulating hormone and metabolite concentrations, and glucose and insulin tolerance were assessed. We also assessed the impact of this protocol on 24 hour profiles of clock and metabolic gene expression in the maternal and fetal liver.

We have previously shown that exposure of pregnant rats to chronic phase shifts of the lighting environment from conception through to early lactation negatively influences a range of metabolic parameters in the subsequent adult offspring [4]. In particular, at 3 months of age, male offspring had increased adiposity and hyperleptinaemia. By 12 months of age, the female offspring also displayed this phenotype, with the addition of hyperinsulinaemia and reduced glucose tolerance and insulin sensitivity. The aim of the current study was to gain an understanding of metabolic and other changes occurring in the pregnant dam throughout gestation caused by CPS exposure which may lead to the programming of metabolic disturbances in the fetus. We found that CPS exposure throughout gestation profoundly altered circadian rhythms of maternal corticosterone, glucose, insulin, leptin, free fatty acids, triglycerides and cholesterol as well as disrupting the timing of food intake and hepatic clock and metabolic gene expression. There was no effect on glucose tolerance, insulin sensitivity or total food consumption in the dams, and yet the rate of maternal weight gain was altered. Furthermore, CPS exposure in utero disrupted the timing of circadian and metabolic gene expression in the developing fetal liver.

The changes in weight gain in the CPS dams were unexpected, being reduced during the first week of gestation. However, as gestation progressed, the CPS dams went on to gain weight more rapidly, such that at the end of gestation they weighed only 4% less than the controls. It is conceivable that the reduction in weight gain observed during the early stages of gestation, a critical stage of embryo development, may have mediated the impact of this protocol on the metabolic health of the offspring. Experimental manipulation of weight gain through moderate caloric restriction during the first half of gestation has previously been shown to influence the metabolic health of rat offspring [6], [7]. Restricting calories to 20% of the control animals from conception to day 13 reduced initial body weight gains, but as gestation progressed and food access returned to ad libitum, the caloric restricted animals caught up so that by the end of gestation there was no significant difference. While this did not affect birth weight, the offspring went on to develop gender dependent hyperphagia, increased adiposity, hyperinsulinaemia and hyperleptinaemia by 5 months of age [6]. This phenotype was programmed through altered neuronal development of the hypothalamic circuits regulating feed intake [7]. It therefore appears that exposure to even moderate nutrient restriction during early gestation can have profound effects on the metabolic homeostasis of the adult offspring. In our study, the CPS protocol did not reduce total food intake at any stage of gestation, but rather changed the timing of food consumption, leading to reduced weight gain during the first week of gestation. This may have been a sufficient insult to program perturbed metabolic homeostasis in the offspring.

At day 20 of gestation, the CPS dams had significantly reduced retroperitoneal fat pad weights and a trend for decreased epigonadal fat weights. Furthermore, maternal liver weight was reduced at the start of the light period, in contrast to the control animals which had elevated liver weights at this time. The reduced fat pad and liver weight was not due to a reduction in these tissues in response to CPS exposure. Instead, the CPS dams failed to increase the size of these tissues in response to pregnancy at the same rate as the controls, as they still had greater fat and liver weights than that of the non-pregnant animals. The reduced liver weights observed at 1200 h may be due to increased hepatic glycogenolysis in response to low plasma glucose, insulin and leptin levels detected through the previous dark period. This is supported by the observation of increased glycogen phosphorylase mRNA in the preceding time points, a key enzyme involved in the release of glucose-1-phosphate from glycogen.

Exposure to the rapid changes in photoperiod has driven changes in the timing of food consumption leading to intermittent grazing rather than consolidated bouts of feeding during the dark period. With each photoperiod shift, the CPS dams are unable to adjust their pattern of food consumption to align with the new phase of darkness, leading to a disrupted profile of food consumption. Repeated exposure to these shifts may have led to decreased food efficiency as reflected in reduced weight gain and adipogenesis, particularly during the early stages of gestation. Weight gain during the later stages of gestation is a reflection of fetal and placental growth, as these tissues account for 25% of maternal weight at term [8]. Given that litter size, fetal weight, and placental weight were all unaffected by the CPS protocol, it is perhaps unsurprising that maternal weight normalised to the controls towards the end of gestation.

An analysis of the metabolic profile of the CPS and control dams at day 20 of gestation reveal profound changes to glucose, insulin, free fatty acids, triglycerides and cholesterol concentrations in plasma, consistent with disrupted timing of food consumption. In the control animals there were strong rhythms of these metabolites, with the exception of plasma triglycerides. However, in the CPS dams, the concentrations of these metabolites were either in antiphase or arrhythmic. Furthermore, the usual strong relationship between glucose and insulin as observed in the control animals was disrupted. Further analysis of the mRNA of some key hepatic enzymes involved in glycolysis and gluconeogenesis suggest a dampened profile of expression. While none of these enzymes were either up or down regulated, the observed arrhythmicity suggests major changes to these metabolic processes.

A rhythm in plasma leptin was observed in the control dams, with high levels at night during the time of feeding, and low levels during the day as previously described in non-pregnant rats [9]. The reduced levels of leptin detected in the CPS dams at night is likely due to the reduced adiposity of these animals, combined with altered timing of food consumption. The junctional zone of the placenta, however, also secretes leptin directly into the maternal blood [10], and therefore we cannot exclude the possibility that the reduced maternal leptin concentrations observed here are due to reduced placental secretion. Leptin plays an important role in fetal development, with its receptor active during neonatal life [11]. In particular, leptin regulates development of hypothalamic circuits regulating energy homeostasis in adult life [12]. Therefore, alterations to the secretion of this hormone from the dam may impact upon the developing offspring, leading to perturbed hypothalamic function. The importance of leptin in neuronal development can be demonstrated by examining ob/ob mice, with offspring showing reduced brain weight and altered projections and synaptic inputs to hypothalamic circuits that regulate feeding [13]. Furthermore, many of these outcomes can be rescued through early postnatal administration of leptin [13]. However, in our model overall maternal leptin levels were reduced by 20% rather than being completely absent. Additionally, unlike ob/ob mice, the offspring retain the ability to secrete their own leptin both in utero and postnatally. Nevertheless, the maternal hypoleptinaemia observed in our study may play a role in the programming of perturbed metabolic homeostasis in the offspring.

Rhythmic melatonin secretion is regulated through indirect neural projections from the suprachiasmatic nucleus (SCN) of the hypothalamus to the pineal [14], and therefore gives an indication of the phase of central SCN rhythmicity. We expected that exposure to rapid photoperiod changes would alter clock functions within the SCN and consequently melatonin secretion. However, melatonin maintained a normal profile of high secretion during darkness and low secretion during light. It has previously been shown that when animals are exposed to either phase delays or phase advances of the photoperiod, the SCN and consequently the timing of melatonin secretion gradually shifts over subsequent days so that eventually the internal clock is in phase with the external light environment [15]. Depending upon the direction and degree of the shift, this can take many days. Therefore, it was surprising to observe that melatonin secretion in the CPS dams had already aligned to the external photoperiod only 1 day after the previous photoperiod reversal. We hypothesise instead that the central SCN clock had never fully adjusted to the photoperiod changes before the lights had been reversed again, and therefore, loosely maintains time with the original photoperiod. Additionally, melatonin secretion of pregnant animals may be less responsive to photoperiod changes than non-pregnant animals. Only through regular sampling throughout the whole protocol will this question be answered. Interestingly, this profile of undisturbed melatonin secretion, despite changes to the timing of light exposure, activity and food consumption is also apparent in human shift workers [16], [17].

Nevertheless, we expect that at stages during the protocol, the melatonin secretion would be suppressed, particularly during the periods of 24 hours of light, as light exposure of sufficient intensity completely suppresses melatonin secretion [18], [19]. Therefore, we cannot rule out the possibility that reduced melatonin secretion plays some role in mediating the changes previously observed in the offspring. Indeed, a recent report has demonstrated the importance of maternal melatonin secretion plays in programming energy metabolism in the offspring [20]. Pups born to dams that were pinealectomised prior to gestation display a similar phenotype to that observed following CPS exposure, including glucose intolerance. Importantly, supplementation of the dams’ nightly drinking water with melatonin, and the concomitant elevation in nocturnal plasma melatonin, attenuated the negative effects of maternal pinealectomy on the offspring. Surprisingly, the negative consequences for the offspring appeared much earlier, with glucose intolerance becoming detectable at 4 months of age, compared to 12 months following the CPS protocol [4].

Compared to the aligned profiles of melatonin secretion in the control and CPS dams, there were large differences in corticosterone secretion between the two groups. Like melatonin, corticosterone secretion is regulated through efferent connections from the SCN. Neurons of the SCN terminate on cells of the PVN, leading to rhythmic CRH release, ACTH secretion from the pituitary and downstream rhythmic secretion of corticosterone from the adrenal (reviewed in [21]). Furthermore, the adrenal response to ACTH secretion also changes over 24 hours, mediated through both local clock mechanisms and SCN-mediated activation of the autonomic nervous system [21], [22]. Together this leads to a rhythmic profile of secretion with increased levels at the end of the light (sleep) period. However, unlike the pineal, the HPA axis is responsive to regulation at many levels via stress, food anticipation and negative feedback mechanisms [23], [24]. Therefore, it was not surprising to observe a disrupted profile of secretion in the CPS dams. The regulating signals driving arrhythmic corticosterone secretion were not identified in this study, but likely reflect altered sensitivity of the HPA axis to stimulation at all levels, as well as changes to the timing of incoming signals.

Regardless of the cause of the disrupted corticosterone secretion, the altered pattern can be expected to mediate important changes in maternal peripheral tissues and the developing fetus. Most peripheral tissues express glucocorticoid receptor (GR), which can then affect the transcription of many genes through both glucocorticoid response elements (GRE) or through interaction with other transcription factors [25]. PEPCK is an example of a gene possessing a GRE [26], the expression of which has been profoundly disrupted in response to CPS exposure. The sensitivity of peripheral tissues also fluctuates over 24 hours through changes in the level of receptor expression and GR-induced transcriptional activity [27], [28]. During development, appropriate glucocorticoid levels play a vital role in normal fetal development [29], with most fetal tissues expressing GR from early embryonic life [30]. The fetus is largely protected from excessive levels of glucocorticoids through the actions of placental 11β-hydroxysteroid dehydrogenase 2 (11β-hsd2), which converts glucocorticoids into their inert forms [31]. Nevertheless, disproportionate levels of glucocorticoids during both early and late fetal development can have profound effects on the programming of adult metabolism [32], [33]. Usually these effects are mediated through reduced growth and subsequent smaller birth weights, although this is not always the case, particularly when exposure occurs during early gestation [34]. While we didn’t observe elevated overall levels of corticosterone in maternal blood, reduced 11β-hsd2 mRNA in placenta, or reduced growth rates of the fetus, it is possible that the disrupted timing of secretion may in some way negatively impact key developmental processes.

Given that the CPS protocol manipulates the dominant zeitgeber to the circadian system (ie light), it was of interest to investigate the impact of this protocol on clock genes in both maternal and fetal tissues. Constant light exposure to pregnant dams has previously been shown to dampen the expression of not only melatonin secretion, but also maternal and fetal clock gene expression, which can be regained through nocturnal melatonin administration [35]. As expected, we found that in the control dams, the expression of Bmal1, Per1, Per2 and Rev-erbα mRNA in the maternal liver was rhythmic, with high amplitude changes in expression from peak to trough. Importantly, the pattern of expression was such that Bmal1 mRNA was in antiphase to Per2, consistent with a functional transcription-translation feedback loop existing in this tissue. When exposed to the CPS protocol however, pregnant dams experienced significant disruption to clock gene expression; the expression of Bmal1, Per2, and Rev-erbα did not significantly fit a sine curve, and the amplitude changes were greatly reduced. Alternatively, within the fetal liver of control animals, Per2 and Rev-erbα mRNA expression was rhythmic with small amplitude changes from peak to trough, but not Bmal1 mRNA expression. Given the importance of Bmal1 in driving rhythmic clock gene expression, and the necessity of this gene in maintaining activity of a transcription-translation feedback loop, it was surprising to then observe robust rhythms of Per2 and Rev-erbα mRNA. Interestingly, CPS fetal livers also rhythmically expressed Rev-erbα and Per2 mRNA, in antiphase to the controls.

What could be driving the rhythmic transcription of these genes, if not the core circadian feedback loop? Schibler and colleagues developed mice with a conditionally active, hepatocyte-specific Rev-erbα transgene, which led to suppression of the core circadian transcriptional feedback loop through suppression of Bmal1 within the liver but not the rest of the body [36]. Under these conditions, circadian expression of the core clock genes was arrhythmic, with the notable exception of Per2, which maintained an identical profile of expression to that of the controls. This suggests that rather than being solely reliant on a functional transcription-translation feedback loop to maintain rhythmicity, Per2 is instead responding to systemic cues. In the context of the fetal liver developing within a rhythmic maternal system, the ability of Per2 to sense or detect maternal rhythms allows fetal tissue to maintain rhythmicity in the absence of an endogenous circadian transcriptional feedback loop. The molecular mechanisms, however, that transmit this information are as yet unknown, but candidates include core body temperature through heat shock factors, maternal and/or fetal glucocorticoids, maternal melatonin and substrate concentrations. Unfortunately, fetal corticosterone concentrations were not assessed in this study, but may have shed some light on this question, particularly given recent demonstrations of the importance of fetal glucocorticoids in conveying maternal rhythmicity to the fetus [35]. Interestingly, we found that the expression of the heat shock protein Hsp105 also changed rhythmically in the control and CPS fetal livers, but with a different phase of expression. This suggests that heat shock factors may mediate the rhythmicity of Per2 and Rev-erbα in the fetus, particularly given the presence of heat shock response elements in the promoter of at least Per2. It cannot be dismissed that disruptions to the timing of circadian gene expression within the developing fetus, as observed here in the fetal liver, may play some role in the programming of adult metabolic disease.

Experiments were funded through an Australian Government National Health and Medical Research Project Grant to DJ Kennaway and TJ Varcoe, APP1009877. DJ Kennaway is funded through a NHMRC senior research fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.