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Endocrinology Vol. 145, No. 11 5364-5372
Copyright © 2004 by The Endocrine Society

Metabolic Consequences of Hypoxia from Birth and Dexamethasone Treatment in the Neonatal Rat: Comprehensive Hepatic Lipid and Fatty Acid Profiling

Eric D. Bruder, Ping C. Lee and Hershel Raff

Endocrine Research Laboratory (E.D.B., H.R.), St. Luke’s Medical Center, Milwaukee, Wisconsin 53215; and Department of Pediatrics (P.C.L.), Department of Pharmacology and Toxicology (P.C.L.), and Department of Medicine (H.R.), Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Address all correspondence and requests for reprints to: Hershel Raff, Ph.D., Endocrinology, St. Luke’s Physician’s Office Building, 2801 West Kinnickinnic River Parkway, Suite 245, Milwaukee, Wisconsin 53215. E-mail: hraff{at}mcw.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neonatal hypoxia is a common condition resulting from pulmonary and/or cardiac dysfunction. Dexamethasone therapy is a common treatment for many causes of neonatal distress, including hypoxia. The present study examined the effects of dexamethasone treatment on both normoxic and hypoxic neonatal rats. We performed comprehensive hepatic fatty acid/lipid profiling and evaluated changes in pertinent plasma hormones and lipids and a functional hepatic correlate, i.e. hepatic lipase activity. Rats were exposed to hypoxia from birth to 7 d of age. A 4-d tapering dose regimen of dexamethasone was administered on: postnatal day (PD)3 (0.5 mg/kg), PD4 (0.25 mg/kg), PD5 (0.125 mg/kg), and PD6 (0.05 mg/kg). The most significant finding was that dexamethasone attenuated nearly all hypoxia-induced changes in hepatic lipid profiles. Hypoxia increased the concentration of hepatic triacylglyceride and free fatty acids and, more specifically, increased a number of fatty acid metabolites within these lipid classes. Administration of dexamethasone blocked these increases. Hypoxia alone increased the plasma concentration of cholesterol and triacylglyceride, had no effect on plasma glucose, and only tended to increase plasma insulin. Dexamethasone administration to hypoxic pups resulted in an additional increase in plasma lipid concentrations, an increase in insulin, and a decrease in plasma glucose. Hypoxia and dexamethasone treatment each decreased total hepatic lipase activity. Normoxic pups treated with dexamethasone displayed increased plasma lipids and insulin. The effects of dexamethasone on hepatic function in the hypoxic neonate are dramatic and have significant implications in the assessment and treatment of metabolic dysfunction in the newborn.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PATHOPHYSIOLOGY OF and adaptation to neonatal hypoxia, a condition associated with serious morbidity and mortality, has been a subject of intense investigation (1, 2, 3, 4). Hypoxemia and the subsequent decrease in O2 delivery to tissues is often due to pulmonary dysfunction, such as bronchopulmonary dysplasia or delayed production of surfactant, or due to cardiac abnormalities (5, 6, 7, 8). There has been much interest in the acute neurological changes associated with neonatal hypoxia, along with the mechanisms of subsequent central nervous system dysfunction in the adult (9, 10, 11, 12). We have shown that hypoxia from birth increases adrenocortical production of corticosterone and aldosterone without a concomitant change in ACTH (13, 14). An additional study has suggested a possible link between adrenal fatty acid composition and the increased production of corticosterone during hypoxia (15). An increase in plasma corticosterone was associated with an increase in plasma lipids such as cholesterol, triacylglycerides (TAG), and free fatty acids (FFA) (16). We have also measured a small increase in plasma insulin concentrations, with no change in plasma glucose, in the hypoxic neonates (17). These changes are consistent with an insulin-resistant state.

The highly potent glucocorticoid dexamethasone is a commonly used treatment for a number of neonatal syndromes, including hypoxia (18, 19). A major motivation for this therapy in hypoxic neonates is the promotion of lung maturation and closure of the ductus arteriosus (20, 21). Dexamethasone has a broad range of action, however, and may lead to unfavorable effects such as left ventricular abnormalities and neurological deficits (22, 23, 24, 25, 26, 27). A well-characterized action of dexamethasone is to modulate hepatic lipid metabolism. In adult rat hepatocytes, dexamethasone inhibits arachidonic acid (20:4n6) synthesis through the modulation of fatty acid desaturase enzymes (28, 29, 30, 31). Dexamethasone also modulates hepatic metabolism of polar and nonpolar lipids, again through alterations in enzyme function (32). A comprehensive characterization of hepatic lipid profiles in the neonate, to our knowledge, has yet to be completed.

The postnatal development of the rat liver has been extensively studied (33, 34). TAG accumulation in the liver is increased during the first week of life (compared with the adult) and is further increased by hypoxia (35, 36). Hepatic glucocorticoid receptor (GR) mRNA expression remains low until the third week of life, coinciding with what has been termed the stress-hyporesponsive period (37, 38, 39, 40). This period is characterized by low plasma concentrations of glucocorticoids and corticosteroid-binding globulin, diminished adrenocortical responsiveness to ACTH, and decreased binding of corticosterone to the GR (37, 41, 42). Dexamethasone appears to have higher affinity for the GR in the 7-d-old rat liver, down-regulating GR expression via intracellular feedback mechanisms (37). Because lipid metabolism in the neonatal liver is under the influence of hormonal factors (28, 29, 30, 31, 32), and neonatal hypoxia increases glucocorticoid production (13, 14, 15), the present study sought to fully evaluate the hepatic effects of hypoxia and/or dexamethasone through lipid profiling.

The goals of the present study were to assess the effects of neonatal hypoxia and/or dexamethasone treatment on plasma concentrations of hormones, lipids and glucose, the hepatic fatty acid and lipid profile, and hepatic lipase activity. We hypothesized that hypoxia induces specific changes in hepatic lipid metabolism that may be modulated by dexamethasone therapy. Newborn rats were exposed to hypoxia from birth to 7 d of age and treated with a tapering dose regimen of dexamethasone to mimic clinical therapy in the human neonate (22). A major advance offered by the present study was the use of comprehensive hepatic lipid profiling, measuring the concentrations of over thirty fatty acid metabolites across 10 distinct lipid classes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal treatment
All experimentation was approved by the Institutional Animal Care and Use Committees of the Medical College of Wisconsin and St. Luke’s/Aurora Sinai Medical Center. Timed pregnant Sprague Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN; n = 16) were obtained at 14 d gestation and maintained on a standard sodium diet (Richmond Standard 5001, Brentwood, MO) and water ad libitum in a controlled environment (lights on, 0600–1800 h). Parturition usually occurred on the afternoon of gestational d 21, during which time rats were kept under observation. After litters were completely delivered, litter size was equalized by cross-fostering and the dam and pups (~13 per litter) were immediately exposed to normobaric hypoxia (12% O2) or kept in room air as control (21% O2) as described previously (16, 43). We have previously shown that this exposure leads to arterial PO2 levels in adults of about 50–55 torr with sustained hypocapnia and alkalosis (43, 44).

Lactating dams were maintained with their litters for 7 d in a hypoxic or normoxic environment (4). Dexamethasone phosphate (Sigma Chemical, St. Louis, MO) was administered sc in a tapering regimen to normoxic and hypoxic pups as follows: postnatal day (PD)3 (0.5 mg/kg), PD4 (0.25 mg/kg), PD5 (0.125 mg/kg), and PD6 (0.05 mg/kg) (22). Control pups were injected with saline. Pups were weighed on each day of injection. At 0800 h on PD7, dams were removed from the chambers. Pups were quickly decapitated, and liver tissue was removed and snap frozen in liquid N2 (n = 4 per treatment).

Lipid profiling
A comprehensive assessment of hepatic lipid profile was performed (Lipomics Technologies, Inc., West Sacramento, CA). Lipids were extracted in the presence of internal standards by the method of Folch et al. (45) using chloroform:methanol (2:1 vol/vol) (8). Individual lipid classes from each extract were separated by preparative thin-layer chromatography as described previously (46, 47). Authentic lipid class standards were spotted on the two outside lanes of the thin-layer chromatography plate to enable localization of the sample lipid classes. Lipid fractions were scraped from the plate and trans-esterified in 3 N methanolic-HCl in a sealed vial under a N2 atmosphere at 100 C for 45 min. The resulting fatty acid methyl esters were extracted with hexane containing 0.05% butylated hydroxytoluene and prepared for gas chromatography by sealing the extracts under N2. Fatty acid methyl esters were separated and quantified by capillary gas chromatography using a gas chromatograph (Hewlett-Packard model 6890, Wilmington, DE) equipped with a 30-m DB-225MS capillary column (J & W Scientific, Folsom, CA) and a flame-ionization detector as described previously (46, 47). Fatty acid ratios were calculated from the sums of each metabolite across all lipid classes. The sums and ratios from each replicated treatment were treated as one datum. Reproducibility was as follows: cholesterol ester (2.0%), diacylglyceride (DAG) (5.5%), FFA (3.5%), lysophosphatidylcholine (LPC) (12.2%), phosphatidylcholine (PC) (5.0%), sphingomyelin (11.4%), phosphatidylethanolamine (PE) (13.0%), and TAG (0.4%).

Hepatic lipase activity
Hepatic tissue was homogenized as described previously (48). The concentration of protein was determined by the method of Bradford (49) (Bio-Rad Laboratories, Hercules, CA). Lipase activity was measured according to Ledford and Alaupovic (50) with some modifications. Briefly, the lipolytic activity was determined by potentiometric titration (at a constant pH of 8.0) of ionized FFA liberated from tributyrin (20 mM) in a citrate-phosphate buffer (1 mM) with 0.01% Triton X-100. Lipase activity was measured in the presence of 0.075 M and 2.4 M NaCl such that both lipoprotein lipase (LPL) and hepatic lipase were evaluated and expressed as total hepatic lipase activity (51). Units are expressed as micromoles of NaOH required to neutralize FFA liberated per gram of protein per minute (n = 8 per treatment).

Plasma measurements
All measurements were performed on pooled samples from each treatment group (3 pups per sample). Insulin was measured by ELISA (Crystal Chem, Inc., Downers Grove, IL). The plasma concentrations of glucose, total cholesterol, and TAG were measured spectrophotometrically as described previously (Pointe Scientific, Inc., Lincoln Park, MI) (16).

Statistical analyses
Fatty acid/lipid profile data obtained were quantitative (nmol fatty acid per gram of tissue) and expressed as mean ± SEM. Because of the complexity of the data set and the increased likelihood of type I error, statistical analyses were performed in two steps. First, significance of differences between normoxic and hypoxic samples of vehicle-treated rats were assessed by unpaired Student’s t tests (P < 0.05). This statistical approach has been validated for this type of metabolomic analysis (46, 47). After this initial screen to explore the data, the interaction of hypoxia and dexamethasone therapy was assessed by two-way ANOVA and Student-Newman-Keuls method for multiple comparisons (P < 0.05). The two-way ANOVA and Student-Newman-Keuls approach was also used to assess differences between normoxic and hypoxic samples not significant by t test, as well as to assess the effects of dexamethasone treatment. Both analytic approaches are presented. Quantitative data were visualized using the Lipomics Surveyor software system. The system creates a heat map graph for significant differences between normoxic and hypoxic samples. The heat map displays statistically significant increases from normoxic values as green squares, and statistically significant decreases as red squares. The brightness of each individual square denotes the magnitude of the difference, as displayed above each of the heat maps. Body mass, plasma measurements, and hepatic lipase activity data were analyzed using two- and three-way ANOVA/Student-Newman-Keuls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of hypoxia and/or dexamethasone treatment on body weight and plasma insulin, glucose, cholesterol, and TAG are shown in Table 1Go. Normoxic and hypoxic pups, as well as normoxic pups treated with dexamethasone, displayed significant increases in body weight from PD3 to PD6. Hypoxic pups treated with dexamethasone showed no increase in body mass and were significantly smaller (PD6) than their untreated counterparts. Hypoxia alone had no significant effect on plasma insulin or glucose. When dexamethasone was administered to normoxic pups, a nearly 10-fold increase in plasma insulin was measured with no change in glucose concentration. Dexamethasone treatment in hypoxic pups also elicited an increase in insulin, but this increase was accompanied by a decrease in glucose. Hypoxia significantly elevated plasma cholesterol and TAG concentrations. Dexamethasone treatment increased plasma TAG and cholesterol in normoxic pups but only increased cholesterol in hypoxic pups.


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  		TABLE 1. Effects of hypoxia from birth and/or dexamethasone (3–6 d of age) on body weight and plasma measurements in the neonatal rat

 
The effects of hypoxia and/or dexamethasone on the concentration of nonphospholipid species in the liver are shown in Fig. 1Go. Hypoxia caused significant increases in hepatic FFA, DAG, and TAG concentrations. The effect of hypoxia was most pronounced for the TAG fraction (3-fold increase). Dexamethasone treatment in normoxic pups had no effect on these lipid classes. However, dexamethasone administration completely blocked the hypoxia-induced increases in hepatic FFA, DAG, and TAG.



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  		FIG. 1. Effect of hypoxia and/or dexamethasone (Dex) treatment on hepatic concentrations of nonphospholipids. The concentrations of FFA, DAG, and TAG were obtained by hepatic lipid profiling (n = 4 per treatment). *, Significant difference from normoxia-vehicle with P < 0.05; #, significant difference from hypoxia-vehicle with P < 0.05.

 
Figure 2Go depicts the effects of hypoxia and/or dexamethasone on hepatic phospholipid concentrations. Hypoxia alone had no effect on the concentration of PC, PE, LPC, or cardiolipin (CL). Likewise, dexamethasone administration to normoxic pups had no effect on these phospholipids. In hypoxic pups, there was a significant effect of dexamethasone on all four lipid classes. The concentrations of PC, PE, and LPC were all decreased in livers from hypoxic pups treated with dexamethasone. In contrast, dexamethasone increased the concentration of CL in the hypoxic liver.



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  		FIG. 2. Effect of hypoxia and/or dexamethasone treatment on hepatic concentrations of phospholipids. The concentrations of PC, PE, LPC, and CL were obtained by hepatic lipid profiling (n = 4 per treatment). #, Significant difference from hypoxia-vehicle with P < 0.05.

 
Figure 3Go shows the concentrations of specific fatty acids in the liver, grouped according to fatty acid family (note: y-axis is a log scale). The data represent the summation of individual fatty acid concentrations in all 10 lipid classes for each treatment group, and these sums are then presented as mean ± SEM. The top panel represents n9 and n7 fatty acids and their saturated precursors. Hypoxia increased the concentrations of 16:0, 18:0, and 18:1n9. Dexamethasone attenuated these increases and also significantly decreased the concentration of 18:0 in the hypoxic liver compared with control. The middle panel depicts the concentrations of hepatic n3 fatty acids. Hypoxia increased the concentration of 18:3, 18:4, 20:5, 22:5, and 22:6 across all lipid classes. All of these increases were attenuated by dexamethasone. The concentrations of four members of the n6 family are shown in the bottom panel of Fig. 3Go. Hypoxia significantly increased 18:2, 18:3, 20:3, and 20:4 concentrations in the liver, and (just as above) dexamethasone attenuated these increases.



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  		FIG. 3. Effect of hypoxia and/or dexamethasone treatment on hepatic concentrations of specific fatty acid metabolites. The upper panel displays the concentrations of metabolites belonging to the saturated and n9 families. Note that 16-1n7, although not an n9 family member, is a product of {Delta}9-desaturation. The middle panel displays metabolites of the n3 family. The lower panel displays metabolites of the n6 family. Fatty acid concentrations were obtained by hepatic lipid profiling (n = 4). The concentration of each metabolite is the sum concentration across all lipid classes measured. Note that the data are presented on a logarithmic scale. *, Significant difference from normoxia-vehicle with P < 0.05; #, significant difference from hypoxia-vehicle with P < 0.05.

 
Figure 4Go shows a heat map illustrating changes in fatty acid metabolite concentrations as measured in distinct lipid classes. The colors indicate the percent change in concentration induced by hypoxia. Colored cells with no number were increased by t test only. Cells marked with a numeral 1 denote a significant increase by hypoxia as well as attenuation by dexamethasone. Cells marked with a 2 denote an increase by hypoxia and attenuation by dexamethasone by ANOVA/Student-Newman-Keuls, but not t, test. A 3 denotes a significant increase by hypoxia (t test only) and a decrease by dexamethasone (ANOVA/Student-Newman-Keuls). Hypoxia significantly increased the concentrations of several fatty acids, mainly in the TAG and DAG fractions. As indicated by the numbers on the heat map, dexamethasone treatment attenuated most of the changes induced by hypoxia. Hypoxia increased all but one of the n3 fatty acids measured in the TAG fraction, and these increases were blocked by dexamethasone. Hypoxia also increased the concentration of 14:0 in the DAG, FFA, LPC, PC, and TAG fractions, as well as the concentration of 22:5n3 (CL, DAG, LPC, PC, PE, and TAG). These increases were again attenuated by dexamethasone.



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  		FIG. 4. Effect of hypoxia and/or dexamethasone treatment on the hepatic concentrations of individual fatty acid metabolites in different lipid fractions. The concentration of each metabolite from each treatment group was used to generate a heat map (n = 4 per treatment). Column headers are the individual fatty acid metabolites for that particular family of fatty acids, and rows are individual lipid classes. The magnitude of the difference in the quantitative data, expressed as percent change from normoxic, is represented by color according to the legend (t test only). Cells marked with a 1 denote a significant increase by hypoxia as well as attenuation by dexamethasone as assessed by t test and ANOVA/Student-Newman-Keuls. Cells marked with a 2 denote an increase by hypoxia and attenuation by dexamethasone (ANOVA/Student-Newman-Keuls). A 3 denotes a significant decrease by dexamethasone (ANOVA/Student-Newman-Keuls). Differences not meeting P < 0.05 by t test are shown in black. Note that triglyceride and diglyceride are equivalent to TAG and DAG, respectively.

 
The effects of dexamethasone on the lipid profile of the normoxic liver are shown in Fig. 5Go. Dexamethasone elicited most of its significant effects in the phospholipid fractions. Dexamethasone had varying effects on fatty acids from the n6 family. The concentration of 20:4n6 was decreased in the CL, PC, and phosphatidylserine (PS) fractions, whereas the concentration of 18:3n6 was increased in the FFA and PE fractions. Dexamethasone also increased the concentration of 20:2n6 in the CL, PC, and PE fractions. Fatty acids from the n3 family were also differentially affected by dexamethasone. The concentration of 22:5n3 was increased in the CL, PC, PE, and PS fractions, whereas 22:6n3 was only increased in the PS fraction. Dexamethasone decreased the concentration of 20:5n3 in both the LPC and PS fractions. Saturated fatty acids, as well as those from the n7 and n9 families, were minimally affected by dexamethasone.



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  		FIG. 5. Effect of dexamethasone treatment on the hepatic concentrations of individual fatty acid metabolites in different lipid fractions. The concentration of each metabolite from each treatment group (normoxia-vehicle vs. normoxia-Dex) was used to generate a heat map (n = 4 per treatment). Column headers are the individual fatty acid metabolites for that particular family of fatty acids, and rows are individual lipid classes. The magnitude of the difference in the quantitative data, expressed as percent change from normoxic, is represented by color according to the legend. Differences not meeting P < 0.05 are shown in black.

 
Hypoxia caused a significant decrease in lipase activity when compared with the normoxic control (210 ± 15 vs. 333 ± 41 µmol fatty acid/g protein·min). A nearly equal decrease in enzyme activity was elicited by dexamethasone treatment (212 ± 12 vs. 333 ± 41 µmol fatty acid/g protein·min). There was no significant additional effect of dexamethasone on hepatic lipase activity in the hypoxic liver.

Calculated ratios of sequential fatty acid metabolites are depicted in Table 2Go. Hypoxia caused a significant decrease in the ratio of the sum of all phospholipids (PE, PS, LPC, PC, CL, and sphingomyelin) to the sum of all nonphospholipids (TAG, DAG, FFA, and cholesterol ester). Hypoxia increased the ratio of TAG to FFA, and dexamethasone attenuated this increase. Across lipid classes, the 18:2n6 to 18:3n6 ratio (an indicator of {Delta}6-desaturase activity) was decreased by hypoxia, and this increase was blocked by dexamethasone. In contrast, dexamethasone decreased this ratio in the normoxic liver. The ratio of 18:2n6 to 20:4n6 (indicator of {Delta}6- and {Delta}5-desaturase activity) was increased by hypoxia, whereas dexamethasone-induced changes were only found for the normoxic liver (increased ratio). Hypoxia also increased the 20:3n6 to 20:4n6 ratio (indicator of {Delta}5-desaturase activity), and dexamethasone was without effect. Last, the ratio of 16:0 to 18:0 (indicator of elongase/{Delta}9-desaturase activity) was increased by hypoxia, and this increase was attenuated by dexamethasone.


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  		TABLE 2. Effects of hypoxia and/or dexamethasone on selected lipid and fatty acid metabolite ratios in the neonatal liver

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of comprehensive lipid profiling has greatly enhanced the ability to characterize changes in lipid metabolism due to a specific disease or treatment (46, 47). The present study used this technology to examine the metabolic effects of hypoxia and dexamethasone treatment on liver function in the neonatal rat. The results reveal metabolic changes that likely play a role in the adaptation to a common neonatal condition (hypoxia), as well as how these changes are affected by an even more common treatment (dexamethasone). Hypoxia elicited significant and specific increases in hepatic lipid and fatty acid concentrations. The major finding of this study was that dexamethasone treatment blocked most of these increases.

Effects of dexamethasone on serum and hepatic lipids in the hypoxic pup
Most individual increases in hepatic fatty acid metabolites due to hypoxia, regardless of lipid class, were blocked by dexamethasone. In fact, the effects of dexamethasone on hepatic fatty acid profiles were greatest in lipid classes most affected by hypoxia, mainly TAG, DAG, and FFA. This was opposite to the effect of dexamethasone in the normoxic liver, where most measured changes were in the phospholipids. Interestingly, dexamethasone decreased the concentration of PC, LPC, and PE in the hypoxic liver, where the effect of hypoxia by itself was not significant. Dexamethasone tended to decrease these phospholipids in the normoxic liver, but the changes were not significant.

There are many possible mechanisms that may explain these findings. One could be modulation of hepatic LPL activity. Dexamethasone treatment increased LPL release in 5-d-old hepatocytes (52). An increase in hepatic LPL release (in combination with increased activity) could increase hepatic FFA concentrations, but these increases were not measured in either normoxic or hypoxic livers after dexamethasone. In addition, we did not measure any change in total hepatic lipase activity in dexamethasone-treated hypoxic pups. However, dexamethasone induced increases in plasma TAG and cholesterol in hypoxic pups, possibly due to inhibition of lipase activity in extrahepatic tissues. LPL gene expression and enzyme activity were decreased in adipose tissue from adult rats treated with dexamethasone (53, 54). Another possible mechanism for the reduction of hepatic TAG in dexamethasone-treated hypoxic pups may be increased synthesis and release of very-low-density lipoprotein (VLDL). Previous studies have found that dexamethasone treatment promotes lipoprotein assembly in the normal neonatal liver (34). The ability to actively secrete TAG-rich VLDL from the liver after dexamethasone treatment is a plausible mechanism and could also partially explain decreased hepatic FFA concentrations. This may also help explain the increase in plasma TAG and cholesterol after dexamethasone treatment to hypoxic pups.

Decreased hepatic FFA concentrations after dexamethasone in hypoxic pups may also be due to up-regulation of fatty acid oxidation. In adult rats, dexamethasone alone or in the presence of increased concentrations of long-chain fatty acids has been shown to stimulate the expression of enzymes involved in peroxisomal ß-oxidation (55). This stimulation was mediated by induction of peroxisomal proliferator-activated receptor (PPAR) mRNA expression (55). In the present study, hypoxia increased the concentration of several fatty acids, all of which may have some stimulatory effect on hepatic PPAR, in particular PPAR{alpha} (56, 57). The administration of dexamethasone may act in a synergistic manner with these fatty acids, up-regulating the processes involved in ß-oxidation.

How do the effects of dexamethasone treatment, and its possible modulation of hepatic PPAR{alpha}, relate to hepatic GR activity? Hepatic GR mRNA expression and receptor number remain low through the first 2 wk of life in the rat, but the GR-ligand complex is biologically active during this time (37). In addition, increased plasma FFA concentrations reduce glucocorticoid binding to hepatic GR and corticosteroid-binding globulin in the immature rat (41, 58). Corticosterone activates PPAR{alpha} and influences key lipid metabolizing enzymes, but this has been measured only in the adult liver (59). We hypothesize that the presence of pharmacological concentrations of dexamethasone, along with increased long-chain unsaturated fatty acids, may activate hepatic PPAR{alpha} in the hypoxic neonate. This activation could stimulate increased fatty acid oxidation and subsequent production of metabolic fuels (e.g. ketones) and could also act to modulate VLDL assembly and secretion.

There may be a number of other mechanisms that may explain the effects of dexamethasone treatment in hypoxic pups, but their mention is beyond the scope of this paper. We are now starting to evaluate each of the most likely potential mechanisms described above.

Effects of hypoxia per se on serum and hepatic lipids
Previous studies have shown that neonatal rats exposed to hypoxia develop hyperlipidemia and increased hepatic lipid content (2, 16, 48, 60). The present results also found increases in plasma cholesterol and TAG and demonstrate that hypoxia-induced fatty liver is, for the most part, due to increased concentrations of hepatic TAG. The nearly 2-fold increase in plasma TAG concentration may be explained by decreased TAG metabolism, because previous work from our laboratory has shown that hypoxia delays maturational increases in hepatic lipase activity (48). Serum TAG in the suckling rat is mainly of intestinal origin, and the neonatal liver can accumulate this TAG, but little is released back into the circulation (34). This is, in part, due to decreased assembly of VLDL in the neonatal liver (34). It is possible that hypoxia causes a further decrease in VLDL synthesis and secretion. This coincides with an increase in TAG concentration in ingested milk from the hypoxic dam (unpublished data).

Increased hepatic TAG synthesis could provide another explanation of the present findings. Although hepatic TAG synthesis in the suckling rat is reduced compared with adults (61), previous studies have shown that, in the adult rat, hepatic phosphatidate phosphohydrolase activity is increased by hypoxia (36). This enzyme is involved in the synthesis of TAG and may be activated as a response to hypoxia in the neonate. Increased incorporation of 16:0 into hepatic TAG has been measured in the hypoxic adult (60), and this change was detected in our neonatal model. The current results are the first, to our knowledge, to measure increased incorporation of other major fatty acids into hepatic TAG during hypoxia. For example, hypoxia increased the concentration of 18:1n9, 18:3n3, and 18:2n6 in the TAG fraction. The accretion of these fatty acids during development is thought to play a pivotal role in energy homeostasis and the structural integrity of membranes (62).

The cumulative concentrations (across all lipid fractions) of most major fatty acids were increased by hypoxia. The concentrations of saturated, n3, and n6 fatty acid metabolites were each increased by hypoxia. The comparison of these concentrations through the use of ratios allows an estimate of fatty acid-metabolizing enzyme activities (63). Hypoxia increased the ratio of 18:2n6 to 20:4n6, indicating that there was an overall decrease in the desaturation of 18:2n6 (63). We also measured an increase in the ratio of 16:0 to 18:0, and a tendency for the 18:0 to 18:1n9 ratio to be decreased (data not shown). Given these results, it is clear that hypoxia alters the metabolism of long-chain fatty acids in the liver.

Effects of dexamethasone per se on serum and hepatic lipids in the normoxic pup
Glucocorticoids have been shown to modulate the activity of a number of desaturase enzymes (28, 29, 30, 31). This characteristic gives this class of steroids a major portion of their antiinflammatory properties, because many mediators of inflammation are 20:4n6-derived prostaglandins and leukotrienes. Previous studies have shown that synthetic and endogenous glucocorticoids depress hepatic {Delta}5- and {Delta}6-desaturase activities in microsomes, leading to decreased production of 20:4n6 (30). Other reports have shown that glucocorticoids also up-regulate the activity of the microsomal {Delta}9-desaturase, the enzyme responsible for the conversion of 18:0 to 18:1n9 and 16:0 to 16:1n7 (29). Both 18:1n9 and 16:1n7 play major roles in hepatic energy production and in the structural integrity of lipids such as PC and PE (62). Interestingly, despite their extensive use, few studies have examined the effects of glucocorticoids on hepatic fatty acid metabolism in the neonate.

Although dexamethasone had no effect on the concentration of any single lipid class, there were significant changes in the fatty acid profiles of most phospholipids. Most notable were decreases in 20:4n6 concentration in the CL, PS, and PC fractions. This agrees with previous measurements of the dexamethasone-induced depression of hepatic {Delta}5- and {Delta}6-desaturase (28, 31). The decrease of 20:4n6 in PC agrees with another study that measured a shift in the fatty acids bound to this phospholipid after dexamethasone treatment (32). Dexamethasone treatment in normoxic pups increased plasma insulin concentrations, with no change in glucose, indicating insulin resistance. Plasma concentrations of TAG and cholesterol were elevated after dexamethasone treatment in normoxic pups. This may be partly explained by the measured decrease in hepatic lipase activity after dexamethasone or by the aforementioned stimulation of hepatic VLDL secretion (34).

Summary
We have used the power of lipid profiling to assess changes in hepatic lipid metabolism in a rat model of neonatal hypoxia. Hypoxia caused specific and significant changes in the fatty acid and lipid concentrations of both the liver and plasma. Using a tapering dose regimen of dexamethasone that mimics what is used in the clinical setting (22), we found that this treatment almost completely blocked the hepatic effects of hypoxia, while augmenting the effects of hypoxia on plasma lipids. We hypothesize that dexamethasone achieves this action, at least in part, through modulation of hepatic PPAR{alpha}, influencing fatty acid oxidation and other lipid-metabolizing processes.

Dexamethasone treatment induced hyperinsulinemia and elevations in plasma lipids associated with an insulin-resistant state. Perinatal hyperinsulinism may predispose animals to diabetes mellitus, obesity, and related cardiovascular risks in later life (64). It remains to be determined whether the benefits of dexamethasone treatment to the hypoxic neonate outweigh the risks these animals may face as adults. Growth restriction due to hypoxia and/or dexamethasone treatment is also an area of concern. Recent studies have suggested that postnatal growth restriction leads to diabetes and the metabolic syndrome, most likely due to modulation of intermediary metabolism (65, 66, 67). A recent study has definitively shown that neonatal dexamethasone treatment leads to long-term decreases in neuromotor and cognitive function (68). To our knowledge, however, the long-term effects of neonatal dexamethasone treatment or hypoxia on intermediary metabolism have not been studied. The clinical implications of the present study therefore generate questions about long-term effects that we are currently exploring.


   Acknowledgments
 
The authors would like to thank Barbara Jankowski, Peter Homar, and Kimberly A. Hagie for their expert technical assistance.


   Footnotes
 
This work was supported by National Institutes of Health Grant DK54685 (to H.R.).

Abbreviations: CL, Cardiolipin; DAG, diacylglyceride; FFA, free fatty acid(s); GR, glucocorticoid receptor; LPC, lysophosphatidylcholine; LPL, lipoprotein lipase; 20:4n6, arachidonic acid; PC, phosphatidylcholine; PD, postnatal day; PE, phosphatidylethanolamine; PPAR, peroxisomal proliferator-activated receptor; PS, phosphatidylserine; TAG, triacylglyceride(s); VLDL, very-low-density lipoprotein.

Received May 7, 2004.

Accepted for publication July 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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