Our study first reveals the organ sizes of the mouse embryo at several time-points during development. These values, calculated in percentage volume, are summarized in Table 2, and are compared with the corresponding organ weight as percent of body weight for the adult 16 week-old mouse [21].

Table 2 Organ-to-body ratio in the SD mouse embryo compared with the adult mouse Full size table

The data reveal that the brain, heart and liver occupy a relatively large percentage of the mouse embryo’s volume at the end of organ development (E13.5). While the percentage volume of the liver decreases slowly towards parturition (E17.5), the corresponding percentages of the brain and heart decrease much more drastically. A comparison with percentage organ weight of an adult 16-week-old mouse reveals that the organ-to-body proportionality continues to decrease significantly post-natally.

Our study also compares how organ growth in the mouse is altered by maternal consumption of a ketogenic diet during pregnancy. Results show an overall increase in volume of the KD embryo at E13.5, but a significant decrease at E17.5, compared with the SD counterpart. At these time points, the KD heart and brain occupied a different percentage volume compared with the SD organs. Additionally, differences revealed by deformation analysis within internal organs were also observed at the two time points, particularly in the pharynx, cervical spinal cord, hypothalamus, thalamus, thymus, mid-brain, pons, left ventricle and in several regions in the liver.

Maternal consumption of KD prior to gestation was found to decrease serum triglycerides in the mouse. This result is similar to that reported by others utilizing the mouse [22]. In contrast, the KD was found to increase triglycerides in the human [23], indicating that the rate of utilization of fat-stores for energy may be different between the two species. However, this needs to be further investigated.

Serum glucose was also found to be reduced, while cholesterol and ketone concentrations were found to be significantly elevated in the KD dams prior to gestation. These changes are as expected during a stable state of ketosis, in which fat intake - including cholesterol intake - is consistently high, and carbohydrate intake is consistently low [2, 24, 25]. All of these metabolites continued to change in the KD dams during pregnancy, with maternal glucose dramatically dropping, while ketone and triglycerides - drastically increasing between E13.5 and E17.5, towards the end of pregnancy. This trend suggests an increase in nutrient demand by the mature fetus, driving maternal metabolism towards high ketosis. Such a state of high ketosis increases the probability of ketoacidosis in the KD dams since any further drop in glucose could trigger more rapid gluconeogenesis, elevating ketones to dangerously high levels, and thus, preventing the utilization of the already-elevated serum ketones. While ketoacidosis was not observed in any of the KD dams during pregnancy, the high ketosis, and particularly the dramatically low maternal blood glucose, could have been the cause of the altered growth exhibited at E17.5. This deleterious effect could have impacted those organs which preferentially use glucose as a source of energy.

While this ketone (β−hydroxybutyrate) can lead to a risky metabolic state, it is in fact a natural product of fat metabolism [26], which also serves as a metabolic fuel [27, 28]. Since maternal ketones easily cross the placenta [29, 30], they should be similarly utilized by the fetus to satisfy its energy requirement during development [27, 28]. If that were indeed the case, then one could naively predict that during a stable, prolonged maternal ketosis, fetal growth would be unaltered, and remain equivalent to that during maternal glycolysis. This, however, neglects to consider the inherent differences between glycolysis and ketosis. While ketones can satisfy energetic requirements, their efficiency as a metabolic fuel is different than that of glucose. In fact, ketones’ efficiency, as measured by oxygen consumption, has been shown to be 25% greater than that of glucose, indicating a significantly greater energy production during ATP hydrolysis [31–33]. Such increased efficiency may then be counteracted by the inhibitory effect of ketones on de novo pyrimidine synthesis, which can slow the rate of cellular growth, as shown in Koehler et al’s in vitro studies (described in [24]). The net result of these opposing actions on embryonic development and fetal growth would then depend on the exact nutrient and energetic demand at any particular developmental phase. Specifically, a positive net effect could explain the increase in embryonic volume, observed at E13.5, yet, a negative – or inhibitory – net effect could, in turn, explain the decrease in embryonic volume, observed at E17.5. Internal organs are potentially also subjected to the same counteracting forces, resulting in altered timing of growth spurts, which could explain the altered KD brain and heart percentage volume at E13.5 and E17.5.

Different effects of KD on organ growth may be explained by organ-specific preference for a particular metabolic fuel. Under non-ketotic conditions, the fetal myocardium has been shown to prefer fatty acids, glucose and lactate over ketones as its major energy substrates [34, 35]. This preference, as measured by substrate utilization rate, was found to primarily depend on relative substrate availability (concentration) [34]. When ketosis was induced by an over-night fast, the neonatal lamb myocardium increased its β OHB oxidation in response to the increase in ketone supply. A similar increase in β OHB oxidation was reported in the brain of the swine fetus following maternal ketosis [36]. In the fetal pig, the brain was also found to increase in weight, protein content and cell size following maternal ketosis [37]. Yet, even independently of maternal ketosis, the developing mouse brain and spinal cord appear to preferentially utilize ketone bodies as an energy source, and as precursors of amino-acid and lipid synthesis [38, 39]. In particular, the rat brain – specifically, the striatum and cortex – were found to have a significantly higher β OHB tissue concentration in late gestation and early postnatal period than at adulthood [40]. Unlike the heart and brain, the fetal liver has been reported to only increase its rate of lipogenesis, but not its rate of β OHB oxidation (i.e. degradation), during maternal ketosis [36]. The potential implication of this is a more rapid triglyceride synthesis, but with a preference for fat deposition in the liver, that could result in an increase in hepatic volume. This, however, was not observed in our KD mice, suggesting the effect of hepatic fat deposition on hepatic volume is insignificant.

Ketone bodies in maternal circulation cross the placenta so that they are available as an energy substrate for fetal use throughout gestation [30]. However, such availability depends on the ketone body carriers, known as Monocarboxylate Transporters (MCTs) [41, 42]. Studies using non-ketogenic gestational diet revealed that the expression of MCT in the placenta decreases at the end of gestation, while the expression of the glucose transporter GLUT1 remains consistently high throughout gestation [29]. This observation suggests that assuming ketolytic enzymes are available to facilitate ketone oxidation [43], then the fetal mouse utilizes both glucose and ketone in early and mid gestation. However, it preferentially utilizes glucose towards the end of gestation, when rapid physiological growth is occuring. In the case of the fetal brain, ketone utilization primarily depends on its concentration in the blood [39]. Hence, prolonged fasting or consumption of a high-fat diet, such as a ketogenic diet, increases the blood-brain barrier’s permeability to ketones [39, 44, 45].

Our observation that the KD embryonic volume as well as myocardium and hepatic percentage volumes were larger than the respective SD ones at E13.5 but not at E17.5 are consistent with the above literature on β OHB oxidation and supply, as modulated by MCT expression. The observation of a localized increase in mid-brain volume in the KD fetus at E17.5, is also in agreement with the studies indicating a strong preference for β OHB as an energy substrate towards the end of gestation [40], as well as with the observation of the increase in fetal brain weight [37]. The high PUFA contents in the KD may result in PUFA accumulation in the brain at the end of gestation, and explain the increased brain volume. This is in agreement with observations by Soares et al. (2009) [46]. The increase in skeletal muscle volume observed at E13.5 may be indicative of increased lipogenesis, while the localized differences indicated by the deformation analysis within internal organs could arise from inhomogeneous energy supply to those sub-regions, or their preference for a different metabolic fuel during their growth.

Another possible cause of the observed differences between the KD and SD embryos may be the inherent difference in macronutrient and micronutrient contents of the respective diets. For example, the KD is high in dietary trans-fatty acids. Studies have indicated a speculative correlation between such high gestational intake of trans-fatty acids and compromised fetal growth and development [47]. In our study, the exact contents of trans fatty-acids was not measured, but may contribute to the observed differences between the KD and SD embryos. Hence, we do not exclude the possibility that some of the observed alterations may be attributed to the differences in micro- and macronutrient intake.

Overall the differences revealed by deformation analysis suggest a deviation from the normal organ development at the end of embryogenesis, which could lead to altered organ function in postnatal life. Specifically, the decrease in relative volume of the right lobe of the thymus, as well as the increase in relative volume in the thalamus, mid-brain, and pons at E17.5 may be indicative of functional changes of those organs. The exact functional effects remain to be elucidated by post-natal studies. Due to the genetic similarity between the mouse and the human, our results could also shed some light on the potential growth effects on the human fetus, if exposed to a similar gestational ketogenic diet.