The most notable finding of this study is that chronic maternal HFD consumption, independent of maternal obesity or diabetes, significantly increased the risk of NAFLD in the developing NHP fetus that persisted into the postnatal period. The evidence for NAFLD includes a 3-fold increase in liver TG levels, activation of several markers of oxidative stress, and premature activation of genes in the gluconeogenic pathway. It has been demonstrated in numerous models, including rodents, sheep, and NHPs, that manipulation of the maternal diet or hormone environment can affect the development of metabolic systems in the offspring (15
). Using NHP models, Nathanielsz and colleagues have demonstrated a broad range of metabolic and developmental defects in the offspring of mothers with nutritional restriction (65
). These broad effects are surprising, because the animals are nutritionally restricted by only 30%. This suggests that primate pregnancies can be affected by subtle changes in nutrition. In the current studies, the animals were fed a diet that derived 35% of its calories from fat and was also high in total calories; this diet is similar to the typical American diet (71
). Several studies in rodents have also investigated the effects of a maternal HFD on the development of metabolic abnormalities in the offspring (21
). While these studies describe a broad range of metabolic abnormalities, only a few describe increased TGs in the liver of adult offspring of pregnant rats fed a HFD; however, none of them determined whether the fatty liver develops prior to or after the development of obesity.
It is well recognized that maternal diabetes, obesity, and even glycemic control within ranges considered normal can markedly affect the developing fetus, thereby increasing the risk of metabolic disease later in life (7
). One of the most important outcomes of this study was the fatty liver phenotype observed in the liver of O–HFD-S and O–HFD-R animals, which suggests that chronic HFD consumption may result in greater lipid transfer to the fetus regardless of maternal obesity. Because of the observational design of our study, we cannot conclude that maternal hyperlipidemia is the sole cause of the adverse outcomes observed; however, such a relationship is plausible. The strong correlation between maternal and fetal glycerol levels suggests that simply increasing maternal lipid as a source of calories in the diet can contribute to changes in the fetus, independent of adiposity or level of insulin resistance in the mother. The increase in liver TGs does not appear to be caused by an increase in de novo lipogenesis, as there was no change in mRNA or protein expression of any of the lipogenic enzymes analyzed (Supplemental Table 3). Indeed, the combination of increased lipid exposure, low fetal insulin, and no change in hepatic enzymes involved in lipogenesis suggests that excess lipid accumulation is a result of increased maternal lipid transfer. Although alterations in glucose metabolism are often considered the primary metabolic adaptations during pregnancy, substantial alterations occur in lipid metabolism as well. In human and rodent pregnancies, there is a shift toward increased lipolysis during the third trimester, and this is thought to be attributable, at least in part, to the normal insulin resistance that occurs during this period in pregnancy. Lipolysis from maternal adipose tissue increases plasma glycerol concentration and elevates fatty acids. Combined with fatty acids from dietary lipids and hepatic TGs, this leads to an increase in TG-rich lipoproteins in maternal circulation available for delivery to the placenta and ultimately provides an increased flux of fatty acids to the fetus (48
). It is possible that insulin resistance in the HFD-fed NHPs may contribute to greater shunting of lipids to the fetus; however, evidence of marked NAFLD was observed in both the O–HFD-S and O–HFD-R animals. Interestingly, fetal liver TG levels were not significantly correlated with maternal or fetal circulating glycerol or TG levels. This suggests that there may be some intrahepatic mechanisms, such as reduced TG export, decreased FA oxidation, or other factors, that increase the risk for steatosis during fetal development. Further study is needed to identify such factors. Furthermore, we recognize that the HFD also has a higher caloric density and more saturated fat; thus it is not possible to definitively identify the major contributor — lipid or total calories — to the fetal lipotoxicity.
One hypothesis for the potential adverse effects of excess lipids on the fetal liver during development relates to the lack of WAT during the early third trimester (36
), which could be used to buffer the excess lipids. In adults, for example, diet-induced obesity overwhelms the WAT stores, and fat accumulates in ectopic organs, such as liver and skeletal muscle, causing insulin resistance. This is illustrated nicely in the “fatless mouse” models as well as in patients with lipodystrophy; in the latter, in the absence of WAT, fat is stored in the liver and muscle, leading to severe insulin resistance (38
). With transplantation of wild-type WAT into this model, lipids dissipate from the liver and deposit in fat pads, and insulin resistance resolves. Therefore, it is possible that as WAT develops during the third trimester in the primate fetus, the fat in the liver will be mobilized and stored in the WAT. However, in O-HFD animals carried to full term and studied at P30 and P180, the fatty liver persisted; although the relative differences were notably less during the postnatal period than during the fetal period, the elevated hepatic TGs were accompanied by a 2-fold increase in percent body fat in O-HFD. The persistence of the fatty liver may not be surprising, given that the offspring are kept with mothers who remain on the HFD and are concurrently breastfeeding. This reduction in liver TG levels could be caused by several factors in the postnatal period, including the development of WAT, but may also be caused by increased physical activity and fuel utilization of the animals as they become independent from their mothers. Juvenile monkeys are very active, especially in group or social housing. Further studies are needed to determine whether the fatty liver persists when O-HFD animals are switched to a low-fat diet during the postnatal period. Studies in rodents have shown that feeding a HFD to pregnant rats throughout pregnancy and during lactation causes a fatty liver phenotype in male offspring that persists into adulthood, even after animals return to a standard chow diet (79
). These rats also displayed abnormalities in the insulin signaling cascade in the liver and were obese and insulin resistant; however, neither the gluconeogenic pathway nor the early development of fatty liver in the fetal or newborn pups was investigated.
One of the primary concerns about lipid accumulation within the liver is lipotoxicity, which can lead to insulin resistance, oxidative stress and/or damage, activation of proinflammatory cytokines, and ultimately liver fibrosis and permanent tissue damage (81
). In the current study, we observed no hyperinsulinemia or any abnormalities in the insulin signaling cascade (either by gene expression or by protein phosphorylation); however, preliminary microarray studies and confirmatory PCR studies identified increased expression of several genes involved in the oxidative stress pathway and proinflammatory cytokines (D. Marks, unpublished observations). The activation of the oxidative stress pathway was confirmed by immunohistochemistry and immunoblot analysis. Furthermore, we also observed elevations in a wide range of inflammatory cytokines in fetal circulation (Supplemental Table 2). Our current understanding of the mechanisms responsible for the development of pediatric nonalcoholic steatohepatitis (NASH) remains limited. Whether this disorder has a fetal component as a result of the marking or “memory” of the liver during fetal life is as yet unknown. The data shown here suggest that the accumulation of excess lipids in the fetal liver is not benign and is associated with increased stress activation (p-JNK) and inflammatory cytokine gene activation, both pathophysiological features found in pediatric patients with NASH. Investigation of the consequences of these histological and biochemical changes on the offspring are underway and may lead to a better understanding of the pathophysiology of pediatric fatty liver disease.
An important question raised by our results and those of others is whether the fatty liver phenotype can be prevented by dietary intervention. In the current study, we chose a straightforward intervention of switching animals that had been on the HFD for more than 4 years (both HFD-S and HFD-R animals) to the low-fat standard chow diet specifically during the fifth-year pregnancy. During the diet reversal protocol, all animals showed a slight improvement of glucose tolerance; however, obese animals remained obese and were still substantially insulin resistant. In spite of this, there was marked improvement, but not complete normalization, in fetal hepatic TGs and partial normalization of the expression of the gluconeogenic enzymes in the O-HFREV group. These results suggest that simply changing to a normal low-fat diet, specifically during pregnancy, can lower, but not eliminate, the risk for fetal hepatic steatosis. Age, parity, or increased prepregnancy BMI could not account for the higher than normal fetal TG accumulation, as the control animals on the standard chow diet had similar numbers of pregnancies, and the effect of the diet reversal was similar in both O–HFREV-S and O–HFREV-R groups. These results imply that a chronic high-calorie, HFD could have long-term effects on maternal/fetal fuel metabolism in future pregnancies. The biological mechanisms through which this HFD during one pregnancy may modify the risk for fetal steatosis in subsequent pregnancies remains unclear. However, the failure of the diet reversal to completely normalize fetal hepatic TGs suggests that the supply or type of endogenous fatty acids may play an important role in addition to the maternal diet. It is well known that maternal obesity and even glucose in the normal range can have a substantial impact on fetal development and health; our current studies did not determine the relative contribution of maternal obesity to the fatty liver phenotype in the offspring. Nevertheless, there were major benefits associated with the diet reversal, which suggests that this is at least a reasonable strategy to help improve the metabolic health of the developing fetus.
Our results also demonstrate that chronic exposure to lipids during pregnancy triggered increased gluconeogenic gene expression associated with PGC1α deacetylation and increased HNF4α expression in the fetal liver, suggesting an important early molecular mechanism by which excess lipids may reprogram hepatic lipid and glucose metabolism in the fetus. The deacetylation of PGC1α has been shown previously to increase PGC1α activity to induce PCK1
transcription and hepatic glucose output in transgenic mice (61
). PGC1α, along with increased HNF4α, is associated with increased PCK1
), and may therefore play an important role in coordinating the premature induction of genes involved in gluconeogenesis that was observed in the O-HFD group. HNF4α can be directly modulated by fatty acyl–CoA derivatives (87
), emphasizing its unique role in transcriptional regulation by nutrients and hormones. Moreover, HNF4α can functionally bind to the promoters of up to 12% of all hepatic genes (89
), suggesting that HNF4α has broad activities and may contribute to a much larger regulatory network of essential metabolic processes in the liver. Although the increases in gluconeogenic gene expression at G130 were relatively small compared with expression in adult liver, in transgenic mice engineered to overexpress hepatic PCK1
at birth, a 2-fold increase was sufficient to induce hepatic glucose production, insulin resistance, and impaired glucose tolerance in adults (69
), suggesting that relatively small increases during development could prove to be significant in the evolution of diabetes. These data would be greatly strengthened by measures of increased hepatic glucose production; however, this was not technically feasible in the NHP fetus. While there could be evidence for greater gluconeogenesis after birth, most studies suggest that this evolves over time, and our animals are in the process of postweaning development. Further studies are currently underway to examine direct binding to the PCK1
promoter and to determine whether increased hepatic glucose production is present during the first year of life in these animals.
In utero adaptations, now referred to as fetal metabolic programming
, were initially documented by Hales, Barker, and colleagues, who described increased cardiovascular mortality and impaired glucose tolerance in association with poor fetal growth (90
). In recent years, the number of babies born large for gestational age has risen dramatically, and along with larger size comes greater risk for developing metabolic syndrome in adulthood (12
). Interestingly, these heavier babies are often born to mothers who are insulin resistant and obese, suggesting a crucial role for maternal overnutrition (33
). Here we illustrated an expanded concern for maternal HFD consumption and risks for NAFLD in offspring independent of maternal obesity or insulin resistance. While the fetuses from HFD-fed NHP mothers weighed slightly less than controls during the early third trimester, at P30 they were normal size and by P90, the animals were significantly fatter (but not heavier), emphasizing the importance of a shift in body composition imposed by early exposure to the HFD.
In summary, our data suggest that exposure to maternal lipid–derived fuels during early pregnancy leads to an excess of lipid accumulation in the liver of the fetal and neonatal offspring (Figure ). Lipid accumulation in the liver is associated with lipotoxicity, likely leading to macrophage infiltration and increased inflammatory cytokine production. This inflammation likely drives the activation of the oxidative stress pathway, which in turn can activate the transcriptional regulators of hepatic gluconeogenesis. Our group has previously demonstrated an increase in heat shock proteins, histone acetylation, and histone deacetylase activity in these fetal livers, suggesting long-term modifications in transcription (93
). Furthermore, offspring had increased adiposity and liver TGs, which indicates that the fatty liver does persist beyond the development of WAT stores. Of great concern is our population of NHP mothers who were neither obese nor insulin resistant prior to pregnancy, but with exposure to a chronic HFD showed a fetal pattern of metabolic abnormalities identical to that of the fetuses from mothers with increased weight gain and insulin resistance. Although our study focused on the fetal liver, it is highly likely that the lipotoxicity is not limited to the fetal liver. Indeed, an increase in aortic fatty streaks has previously been observed in human fetuses from mothers with chronically or acutely elevated cholesterol, despite the lack of correlation between maternal and fetal cholesterol levels (94
). Our findings of increased fetal steatosis and subsequent adiposity support what we believe to be a previously unrecognized risk of fetal programming of obesity resulting from early exposure to excess maternal lipids in utero. Finally, the current dietary guidelines for lowering blood glucose in pregnancy include substituting lipids for carbohydrates (95
); however, the results of the current study emphasize the need to carefully balance carbohydrate and fat intake during pregnancy to reduce the risk of both hyperglycemia and hyperlipidemia during this critical developmental phase. Failure to recognize that maternal diet and maternal obesity play a critical role in fetal programming of adult disease may ultimately lead to an acceleration in the obesity epidemic through successive generations, independent of further genetic or environmental factors (96
Effects of increased maternal/fetal lipid supply on hepatic lipotoxicity in the fetus.