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Fetal adaptations to periods of substrate deprivation can result in the programming of glucose intolerance, insulin resistance, and metabolic dysfunction in later life. Placental insufficiency can be associated with either sparing or sacrifice of fetal liver growth, and these different responses may have different metabolic consequences. It is unclear what intrahepatic mechanisms determine the differential responses of the fetal liver to substrate restriction. We investigated the effects of placental restriction (PR) on liver growth and the hepatic expression of SLC2A1, IGF1, IGF2, IGF1R, IGF2R, PPARGC1A, PPARA, PRKAA1, PRKAA2, PCK2, and HSDL1 mRNA in fetal sheep at 140–145 days of gestation. A mean gestational arterial partial pressure of oxygen less than 17 mmHg was defined as hypoxic, and a relative liver of weight more than 2 SD below the mean liver weight of controls was defined as reduced liver growth. Fetuses therefore were defined as control-normoxic (C-N; n = 9), PR-normoxic (PR-N; n = 7), PR-hypoxic (PR-H; n = 8), or PR-hypoxic reduced liver growth (PR-H RLG; n = 4). Hepatic SLC2A1 mRNA expression was highest (P < 0.05) in the PR-H fetuses, in which liver growth was maintained. Expression of IGF1 mRNA was decreased (P < 0.05) only in the PR-H RLG group. Hepatic expression of HSDL1, PPARGC1A, and PCK2 mRNA also were increased (P < 0.05) in the PR-H RLG fetuses. The present study highlights that intrahepatic responses to fetal substrate restriction may exist that protect the liver from decreased growth and, potentially, from a decreased responsiveness to the actions of insulin in postnatal life.
A range of epidemiological, clinical, and experimental studies have highlighted that whereas fetal adaptations to periods of substrate deprivation may enhance fetal survival, they also can result in the programming of insulin resistance, glucose intolerance, and type 2 diabetes in later life . It has been proposed that when the fetal nutritional environment is suboptimal, adaptive responses result in development of the “thrifty phenotype,” which involves a relative decrease in overall body growth and in the growth of organs such as the gut and liver but differential sparing of the growth of key organs such as the brain and heart . It also has been proposed that the metabolic adaptations resulting in the thrifty phenotype are designed to enhance survival when postnatal nutrition is poor but then become detrimental when postnatal nutrition is more abundant than that experienced in the prenatal environment.
In the rat, intrauterine growth restriction (IUGR) induced by bilateral uterine artery ligation during late pregnancy results in fasting hyperglycemia and hyperinsulinemia in adult life . Hepatic peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A; also known as PGC1A) is increased in the livers of the offspring at birth and at 21 days of life . PPARGC1A is a transcriptional coactivator of nuclear receptors that control the hepatic expression of key gluconeogenic enzymes, including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 2 (PCK2; also known as PEPCK) . An increase in the mRNA levels of the gluconeogenic enzymes also was observed in the liver at birth and on Postnatal Day 21 in growth-restricted animals, whereas hepatic glucokinase mRNA levels were significantly decreased .
A significant effect of a low-protein diet during pregnancy and lactation on hepatocyte proliferation, liver growth, and morphology in the offspring has been observed as well [6, 7]. In addition, a loss of the initial insulin suppression of glucose output in vitro and an associated increased activity of hepatic PCK2 has been reported .
In rats, administration of dexamethasone during the third week of pregnancy results in a permanent upregulation of hepatic PCK2 mRNA expression and activity . It has been proposed that maternal or fetal glucocorticoids may be implicated in the prenatal programming of glucose intolerance after prenatal substrate restriction .
These studies highlight the vulnerability of hepatic development to periods of undernutrition in a species for which organogenesis and rapid cellular proliferation occurs during late gestation and early postnatal life. Relatively few studies, however, have investigated the impact of poor fetal substrate supply on hepatic development in species such as the sheep or human, in which organogenesis and the period of rapid hepatocyte proliferation and liver growth occurs during earlier pregnancy. We have used a model of placental and fetal growth restriction in the sheep, in which the majority of uterine endometrial caruncles are excised in the nonpregnant ewe . After conception, placental growth and function are restricted, resulting in chronic fetal hypoxemia and hypoglycemia and in fetal growth restriction during late gestation [10–12]. In this model, the extent of fetal growth restriction is determined by the number of remaining placental attachment sites and the degree to which compensatory growth occurs in each placentome during pregnancy . Using this model, we have demonstrated that whereas relative fetal liver weight tends to decrease as fetal weight decreases, the liver growth response in placentally restricted (PR) fetuses is variable, with relative liver weights ranging between 12 and 25 g/kg at a fetal body weight of approximately 3 kg in late gestation (term, 150 ± 3 days of gestation) . It is not known whether specific intrahepatic responses determine the extent to which liver growth is sacrificed when fetal substrate supply is restricted. We hypothesize that poor liver growth in the IUGR fetus may result from a decreased expression of the intrahepatic glucose transporter, SLC2A1, and of IGF1. We also hypothesize that when liver growth is reduced, a specific upregulation occurs in the expression of PPARGC1A and the gluconeogenic enzyme, PCK2. We therefore have determined whether differences exist in hepatic expression of the solute carrier family 2 (facilitated glucose transporter) member 1, SLC2A1 (previously known as GLUT1); the intracellular fuel sensor, protein kinase, AMP-activated, alpha 1 catalytic subunit (PRKAA1) and protein kinase, AMP-activated, alpha 2 catalytic subunit (PRKAA2); the 11β-hydroxysteroid dehydrogenase type 1 isoform (HSDL1), which converts cortisone to cortisol; and the growth factors IGF1 and IGF2 and their receptors, IGF1R and IGF2R, in hypoxic PR fetuses in which liver growth was either maintained or decreased. We also have investigated whether the hepatic expression of factors that regulate glucose metabolism and fatty acid oxidation (PPARGC1A, PCK2, peroxisome proliferator-activated receptor alpha [PPARA], and glycerol-3-phosphate dehydrogenase 1 [soluble] [GPD1]) is different when fetal liver growth is either maintained or decreased in hypoxic PR fetal sheep during late gestation.
All procedures were approved by the Animal Ethics Committee of the University of Adelaide (Adelaide, South Australia, Australia). Ewes were housed in individual pens in rooms with a 12L:12D photoperiod. Pregnant ewes were fed once daily at 1100 h with 1 kg of lucerne chaff (85% dry matter [DM]; metabolizable energy content, 8.3 MJ/kg DM) and 0.3–0.5 kg of concentrated pellets containing straw, cereal, hay, clover, barley, oats, lupins, almond shells, oat husks, and limestone (90% DM; metabolizable energy content, 8.0 MJ/kg DM; Johnson & Sons). The diet provided 100% of the energy requirements for the maintenance of a pregnant ewe as specified by the Ministry of Agriculture, Fisheries, and Food (UK). Food and water intake was monitored daily in all ewes, and the amount of feed not consumed over a 24-h period was recorded. No difference in feed intake was found between ewes in the control cohort and those that underwent the carunclectomy surgery.
Twenty-eight Merino ewes between 140 and 145 days of gestation were used in the present study. In 19 ewes (PR group), carunclectomy was performed to remove the majority of the endometrial caruncles (49.2 ± 1.7 per horn) from the uterus of the nonpregnant ewe before mating, leaving approximately 4.9 ± 0.2 caruncles per horn as described previously [11, 13]. Following the carunclectomy surgery, the ewes were monitored for 4–7 days and were mated after approximately 10 wk. Ewes in the control (n = 9) and PR (n = 19) groups were transported to the animal holding facility, and surgery was performed between 103 and 117 days of gestation under aseptic conditions as described previuosly [11, 13]. Briefly, general anesthesia was induced and maintained with 4% halothane in O2, and catheters were inserted into a fetal jugular vein, carotid artery, and amniotic fluid. In ewes carrying twin fetuses, only one fetus was catheterized. Fetal catheters were exteriorized through an incision made in the ewe's flank. Animals were allowed to recover from surgery for at least 4 days before blood sampling commenced. Fetal arterial blood (0.5–3.5 ml) was collected routinely to assess blood gas and pH status of the fetus (ABL 520 Blood Gas Analyzer; Radiometer). In a subgroup of fetuses, plasma samples, stored at −20°C, were available for the determination of plasma glucose concentrations during late gestation.
Postmortem examination was performed between 140 and 145 days of gestation. Fetal weight was recorded, fetal organs removed and weighed, and samples of the fetal liver snap-frozen in liquid nitrogen and stored at −80°C until analysis.
Total RNA was extracted from fetal liver samples as described previously , and total RNA was purified using the RNeasy Mini Kit (Qiagen) as recommended by the manufacturer. Total RNA was quantified by spectrophotometric measurements at 260 and 280 nm. Complementary DNA was synthesized from 5 μg of total RNA using Superscript III (Invitrogen) by reverse transcription (RT) . Controls containing no RNA transcript or no Superscript III were used to test for genomic DNA contamination.
The relative abundances of SLC2A1, PRKAA1, PRKAA2, HSDL1, IGF1, IGF2, IGF1R, IGF2R, PPARGC1A, PCK2, GPD1, and PPARA mRNA transcripts in fetal liver were measured by quantitative RT-PCR using SYBR Green Master Mix in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with ovine-specific primers validated to generate a single transcript as confirmed by the presence of a single double-stranded DNA product of the correct size (Table 1) and sequence as confirmed in turn using a Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Each quantitative RT-PCR well contained 5 μl of Power SYBR Green Master Mix (Applied Biosystems), 1 μl each of forward and reverse primer (GeneWorks) for the appropriate gene (Table 1), 2 μl of water, and 50 ng/μl of cDNA (1 μl) to give a total volume of 10 μl. Controls for each primer set containing no cDNA were included on each plate. Three replicates of cDNA from each liver sample were performed for each gene on each plate, and each plate was repeated in duplicate to ensure a consistent result. Amplification efficiencies were determined from the slope of a plot of Ct (defined as the threshold cycle with the lowest significant increase in fluorescence) against the log of the cDNA template concentration (range, 1–100 ng/μl). The Ct values were in the linear amplification range (~16–26 cycles) for all genes. The abundance of each transcript relative to the abundance of the reference gene ribosomal protein, large, P0 (RPLP0; also known as RPP0) was calculated using Q-Gene analysis software .
Plasma glucose concentrations were determined during late gestation in a subset of animals for which plasma samples were available (control, n = 8; PR, n = 9). Plasma glucose concentrations were measured by enzymatic analysis using hexokinase and glucose-6-phosphate dehydrogenase to measure the formation of NADH photometrically at 340 nm (Konelab 20, Program Version 6.0 automated analysis system; Thermo Fisher Scientific). The sensitivity of the assay was 0.5 mmol/L, and the intra- and interassay coefficients of variation were both less than 5%.
All data are presented as the mean ± SEM. Placental restriction resulted in three experimental groups as defined by the level of fetal arterial partial pressure of oxygen (PO2) and by either sparing or reduction in fetal liver growth. A mean gestational arterial PO2 of less than 17 mmHg was defined as hypoxic, and a relative fetal liver weight more than 2 SD below the mean relative liver weight of control fetuses (i.e., <16.4 g/kg) was defined as reduced liver growth. The three experimental groups therefore included fetuses categorized as PR-normoxic (PR-N; n = 7), PR-hypoxic (PR-H; n = 8), and PR-hypoxic reduced liver growth (PR-H RLG; n = 4). All control fetal sheep were normoxic (C-N; n = 9). Differences in fetal weight, relative liver weight, mean gestational PO2, arterial partial pressure of carbon dioxide (PCO2), pH, oxygen saturation, hemoglobin, mean plasma glucose, and hepatic gene expression between groups were determined by a one-way ANOVA using the Statistical Package for Social Scientists (SPSS) for Windows (Version 14; SPSS, Inc.). Relationships between variables were determined by linear regression using SigmaPlot 10.0 (SPSS, Inc.). A probability level of 5% (P < 0.05) was taken to be significant.
Fetal weight was significantly lower in the PR-N group when compared to the C-N fetal sheep and was significantly lower in the PR-H and PR-H RLG groups when compared to either the C-N or PR-N group (P < 0.001) (Fig. 1A). Mean gestational arterial PO2 also was significantly lower in the PR-H and PR-H RLG groups when compared to either the C-N or PR-N group (P < 0.001) (Fig. 1B). A direct correlation was found between fetal weight and mean gestational PO2 when data from all fetal sheep were combined (y = 0.21x + 0.5, r2 = 0.67, P < 0.0001). Arterial oxygen saturation was significantly lower in the PR-H and PR-H RLG groups when compared to the C-N and PR-N groups (P < 0.001), and fetal arterial hemoglobin concentration was significantly higher in the PR-H and PR-H RLG groups when compared to the C-N and PR-N groups (P < 0.05) (Table 2). No differences were found in either fetal arterial PCO2 or pH between the four groups throughout late gestation (Table 2).
No difference was found in relative fetal liver weight between the C-N, PR-N, and PR-H groups (Fig. 1C). Mean relative fetal weight was significantly lower, however, in the PR-H RLG group when compared to the other fetal groups (P < 0.005) (Fig. 1C). Plasma glucose concentrations were significantly lower in the PR-H group than in the PR-N group and were lower in the PR-H RLG group when compared to the C-N and PR-N groups (C-N, 0.85 ± 00.04 mmol/L; PR-N, 0.97 ± 0.05 mmol/L; PR-H, 0.66 ± 0.13 mmol/L; PR-H RLG, 0.47 ± 0.05 mmol/L; P < 0.005).
No difference was found in hepatic PRKAA1 or PRKAA2 mRNA levels between the four groups (Table 3). Hepatic expression of PRKAA2 mRNA was approximately 30- to 50-fold higher than that of PRKAA1 mRNA in all fetal groups (Table 3). Hepatic SLC2A1 mRNA expression was higher (P < 0.05) in the PR-H group in which liver growth was maintained when compared to the C-N group, and no difference was observed in the level of SLC2A1 mRNA expression between the C-N, PR-N, and PR-H RLG groups (Fig. 2A). HSDL1 mRNA expression was significantly higher (P < 0.05) in the PR-H RLG group when compared to the C-N, PR-N, and PR-H groups (Fig. 2B).
Hepatic expression of IGF1 mRNA was significantly lower (P < 0.05) in fetuses of the PR-H RLG group when compared to fetuses of the C-N and PR-N groups (Fig. 3). No difference, however, was found in the hepatic expression of IGF2, IGFI2, or IGF2R mRNA between the four treatment groups (Table 3).
The PPARGC1A mRNA expression was higher (P < 0.05) in the livers of the PR-H RLG group when compared to those of the C-N group, and hepatic PCK2 mRNA expression also was higher (P < 0.001) in the PR-H RLG group when compared to the other three groups (Fig. 2, C and D). Whereas hepatic PPARA mRNA expression was highest in the PR-H RLG group, no significant difference was observed in PPARA expression (P = 0.07) between the groups. Similarly, whereas GPD1 mRNA expression was lowest in the PR-HLG group, no significant difference was found in GPD1 expression (P = 0.07) between the groups (Table 3).
A significant positive relationship was found between hepatic PPARA and PPARGC1A mRNA expression (y = 0.631x – 0.004, r2 = 0.50, P < 0.001) and between plasma glucose and hepatic GPD1 mRNA expression (y = 0.062x − 0.009, r2 = 0.36, P < 0.05) when data from all groups were combined.
In the present study, we have demonstrated that hepatic SLC2A1 mRNA expression is increased in PR, hypoxic fetuses in which liver growth was maintained during late gestation. In these fetuses, no upregulation of hepatic HSDL1, PPARGC1A or PCK2 mRNA expression and no decrease in hepatic IGF1 mRNA expression occurred. These results indicate that a subset of hypoxic PR fetuses may have specific intrahepatic mechanisms ensuring that hepatic IGF1 expression and liver growth are maintained. In contrast, in those PR hypoxic fetuses in which liver growth was decreased, an upregulation of HSDL1, PPARGC1A, and PCK2 mRNA expression and a decrease in hepatic IGF1 mRNA expression occurred when compared to normoxic control or hypoxic PR fetuses. These differential patterns of hepatic growth and gene expression in PR hypoxic fetuses are of potential importance in determining whether there will be programming of glucose intolerance in postnatal life.
Whereas induction of placental growth restriction by experimental reduction of the number of available placental attachment sites from conception results in a decrease in placental growth and function, the extent of the placental and, hence, fetal growth restriction is determined by the extent of the compensatory growth response of the remaining placentomes . The present study included a group of PR fetal sheep in which arterial PO2 and plasma glucose concentrations were not different from those of control fetuses, and these PR fetuses also had body weights and relative liver weights comparable to those of the control fetuses during late gestation. As expected based on previous studies, experimental PR also resulted in fetuses that were chronically hypoxemic, hypoglycemic, and growth restricted when compared to either the C-N or PR-N groups. Interestingly, the present study also included a subset of hypoxemic PR fetuses in which fetal liver growth was maintained and a second subset in which fetal liver growth was significantly reduced. These differences in fetal liver growth did not appear to be explained by the degree of fetal hypoxemia or by the prevailing oxygen saturation, which were the same in both PR-H groups. In addition, no difference in plasma glucose concentrations was found between the PR-H groups in which liver growth was either maintained or decreased. Systemic measures of placental growth restriction such as arterial PO2 and plasma glucose concentrations, however, may not directly reflect the substrate environment of the hepatocyte. In fetal sheep, under normal conditions, two thirds of umbilical blood flow supplies the liver (equivalent to ~70% of total hepatic blood flow), with about one third passing through the ductus venosus . It has been demonstrated previously in the sheep that hypoxemia increases shunting of blood through the ductus venosus, most probably to ensure an increase in oxygen and glucose supply to key fetal organs such as the heart and the brain . In experimental studies in which a stent was inserted to dilate the ductus venosus in the late-gestation sheep fetus, liver blood flow and relative liver weight were each decreased, and this was associated with a decrease in hepatic cell proliferation . It therefore is possible that in the current study, liver growth was maintained in a subset of PR-H fetuses because this group had less shunting of blood through the ductus venosus than occurred in the subset of fetuses in which liver growth is reduced. This could potentially explain the differential pattern of liver growth in the PR-H fetuses, but it is not clear why there would be differential shunting of blood flow through the ductus venosus when the PR-H and PR-H RLG groups had no difference in the level of fetal hypoxemia.
An alternate explanation for the differential pattern of liver growth in the PR-H fetal sheep may be that an intrahepatic response to fetal substrate restriction acts to maintain fetal liver growth, and that this response is not present among the group of fetuses in which liver growth is reduced. It may be relevant in this context that the hepatic expression of SLC2A1 mRNA was highest in the PR-H group in which liver growth was maintained. SLC2A1 is the predominant fetal glucose transporter isoform that mediates basal glucose transport into rapidly growing cells, and SLC2A1 has been expressed in relatively higher concentrations in all fetal tissues examined when compared with the adult [18–21]. It has been shown previously that bilateral uterine artery ligation in the pregnant rat results in fetal growth restriction and an uncoupling of hepatic cellular energy and redox states, resulting in less ATP generated per unit of glucose . These changes also were associated with enhanced SLC2A1 mRNA levels in the liver of the fetal and neonatal rat after utero-PR in late pregnancy . In the present study, no difference was found in the expression of the either the catalytic isoforms (alpha 1 or alpha 2 catalytic subunit) of PRKAA in the fetal liver in the PR-H or C-N groups, but this may not reflect changes in the phosphorylation status or activation of this intracellular fuel sensor. One possibility is that when SLC2A1 mRNA expression does not increase significantly in the liver of the hypoglycemic, growth-restricted fetus, the lack of a compensatory increase in hepatic glucose uptake leads to a decrease in hepatic IGF1 mRNA levels, with a consequent decrease in liver growth. Interestingly, in rats exposed to a low-protein diet throughout pregnancy, a decrease in hepatocyte proliferation and in IGF1 production also occurs in the fetal liver and at 3 mo of age, and the livers of these offspring have fewer but larger lobules, with a concomitant loss of insulin suppression of glucose output in vitro [7, 24].
In the present study, we also found that hepatic expression of HSDL1 and PCK2 mRNA was increased in the hypoxic PR fetuses in which liver growth was decreased, but not in the hypoxic PR fetuses in which liver growth was maintained. It recently was demonstrated that hepatic PCK2 mRNA expression increased following the induction of chronic hypoglycemia in fetal sheep, and that this was associated with a decrease in circulating insulin and an increase in plasma cortisol . Thus, one explanation of the findings in the present study is that in the subset of hypoxic, hypoglycemic, growth-restricted fetuses in which liver growth is sacrificed, low hepatic glucose uptake results in the increased expression of HSDL1, the enzyme that converts cortisone to cortisol in the hepatocyte. Cortisol has been shown to play a key role in the upregulation of hepatic PCK2 activity and expression in the late-gestation fetus [26, 27], so the increase in hepatic HSDL1 mRNA expression and intrahepatic cortisol production may stimulate increased PCK2 mRNA expression.
The PPARGC1A mRNA expression was higher in the fetal livers of the PR-H RLG group when compared to those of the C-N but not the PR-N or PR-H groups. Similarly, after bilateral uterine ligation in late gestation, an increase in hepatic PPARGC1A expression occurs in the growth-restricted rat fetus . Whereas no significant difference was found in hepatic PPARA mRNA expression between the groups in the current study, a positive relationship was observed between PPARGC1A and PPARA mRNA expression across all groups. Thus, it appears that the consequences of a decrease in liver growth in the growth-restricted sheep fetus are more likely to be associated with differences in hepatic glucose, rather than fatty acid, metabolism.
In summary, the present study has demonstrated that restriction of placental function and fetal growth restriction can result in different patterns of fetal liver growth and hepatic gene expression. One subset of hypoxic, hypoglycemic, IUGR fetuses has a downregulation of hepatic IGF1 mRNA expression, decreased liver growth, and upregulation of HSDL1 and PCK2 expression. In a second subset of IUGR fetuses, however, fetal liver growth is maintained, and in those fetuses, an increase in SLC2A1 expression but no decrease in hepatic IGF1 expression or changes in HSDL1 and PCK2 expression is found. These differential patterns of hepatic growth and gene expression are of potential importance in determining whether programming of glucose intolerance will occur in postnatal life. It has been demonstrated in the rat that bilateral uterine artery ligation during late gestation results in fetal hypoglycemia, upregulation of hepatic gluconeogenic enzyme expression, and impaired glucose tolerance from as early as 1 wk after birth [3, 28, 29]. Similarly when the fetal rat is exposed to a low protein diet during late gestation, there is a decrease in fetal liver growth and an upregulation in the expression of hepatic gluconeogenic enzymes and impaired glucose tolerance in postnatal life [7, 8]. In the rat, the fetal liver undergoes a rapid period of cellular proliferation during late gestation and may be more vulnerable to the effects of substrate deprivation than the fetal liver in the sheep or human during late pregnancy. The present study highlights that unlike the rat, intrahepatic responses to fetal substrate restriction may occur in the sheep and, potentially, the human that protect the liver from decreased growth and, potentially, from a decreased responsiveness to the actions of insulin in postnatal life.
We wish to acknowledge Lisa Edwards, Anne Jurisevic, and Laura O'Carroll for their assistance with surgical procedures. We also are grateful to Bernard Chuang for his assistance with the molecular analyses reported in the present study.
1Supported by a Program Grant Award from the National Health and Medical Research Council of Australia (ICMcM) and through a Project Grant in Aid from Channel 7 Children's Research Foundation (ICMcM). J.L.M. was supported by a Postdoctoral Fellowship from the National Heart Foundation of Australia (PF 03A 1283).