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Department of Pediatrics (K.-D.N., J.D., M.R., W.R.) and Department of Experimental Medicine I (K.-D.N., H.S.), Nikolaus Fiebiger Centre of Molecular Medicine, University of Erlangen-Nuernberg, 91054 Erlangen, Germany; and Experimental Neonatology (H.S.), Department of Pediatrics, Medical University of Innsbruck, 6020 Innsbruck, Austria
Address all correspondence and requests for reprints to: Kai-Dietrich Nüsken, M.D., Department of Pediatrics, Friedrich-Alexander-University Erlangen-Nuernberg, Loschgestrasse 15, 91054 Erlangen, Germany. E-mail: nuesken{at}yahoo.com.
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Introduction |
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Current research on this model of uteroplacental insufficiency focuses on the phenomenon of metabolic programming by the altered intrauterine environment and its impact on the postnatal development (12, 13). LIG in the study group to be compared with SOP control animals has been preferred to the model of unilateral uterine artery ligation with internal controls (fetuses of the unligated uterine horn), because hyperperfusion of the unligated horn is supposed to result in increased fetal growth with possible metabolic consequences (4). However, it has not been investigated yet whether sham operation by itself may cause metabolic programming, because the intrauterine milieu is affected at least temporarily by surgery and recovery.
The adipocytokine visfatin, which is expressed in bone marrow, liver, muscle (14), and adipose tissue of mice and humans, has been reported to promote anabolic effects in vitro (15). There are controversial results concerning the association of visfatin with obesity and diabetes (15, 16). However, data on visfatin in the LIG model are not yet available.
This study focused on the hypothesis that not only bilateral uterine artery ligation but also sham operation may result in IUGR and its potential metabolic sequelae. Therefore, we examined offspring of LIG, SOP, and untreated control dams. Additionally, we investigated serum visfatin as well as visfatin expression in liver, muscle, and adipose tissue and its potential contribution to impaired glucose tolerance subsequent to IUGR. Remarkably, we have found that sham operation may result in both significant IUGR and metabolic programming.
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Materials and Methods |
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Time-mated female Wistar rats (HsdCpb:WU) in their first pregnancy were purchased from Harlan-Winkelmann (Borchen, Germany) and housed individually from d 13 postcoitum (pc) under air-conditioning providing a temperature of 22 ± 2 C, constant relative humidity of 55%, and a 12-h light, 12-h dark cycle. The animals were allowed free access to standard rat chow and water. On d 19 p.c., the pregnant rats were anesthetized by im injection of midazolam (5 mg/kg) and ketamine (100 mg/kg). After midline laparotomy, both uterine horns were pulled out of the abdomen until exposed completely, and the living fetuses were counted. Next, either a ligation of both uterine arteries (LIG) at the most caudal point accessible was carried out with 6–0 Prolene (Ethicon, Norderstedt, Germany), or the suture material was not fixed but removed after identical anesthetic and surgical procedures (SOP). All animals carried three to nine living fetuses per uterine horn (five to seven in the LIG group). The uterus was then placed back into the abdominal cavity that was closed with a 4–0 Vicryl suture (Ethicon). All surgeries were performed between 1100 and 1400 h. The rats started drinking and taking food within 4–8 h and recovered fully before the beginning of the dark cycle at 1900 h. Untreated rats served as controls. All pregnant animals delivered spontaneously on d 21 or 22 pc within a time frame of 12 h. Except for three fetuses of one of the SOP dams, all fetuses survived the procedure.
Six male LIG pups (two litters, birth weight between 4.3 and 5.2 g) and 12 male SOP pups (four litters, birth weights 5.9–6.4 g) were chosen according to their birth weight (smallest six males available in the LIG litters; heaviest 12 males available in the SOP litters). Six male control pups (one litter, birth weights 6.6–7.2 g) were the only males available in the respective litter. Our selection criteria are based on the finding that only LIG fetuses in the caudal position (near the ligation) show growth restriction and should be compared with SOP fetuses in the caudal position, which are most often the heaviest pups of the respective litter (17). Each litter was reduced to six male pups immediately after birth to assure uniformity of the litter size and optimal access to milk during nursing. All litters were transferred to untreated foster mothers, whose own pups had been killed, and remained with them until weaning. One foster mother cared for all the LIG pups, two foster mothers raised the SOP subjects, and one foster mother hosted all the control pups. After weaning at d 28 of life, the rats were kept in units of two animals with free access to standard rat chow and water.
Clinical and paraclinical examinations of the offspring
LIG, SOP, and control offspring were weighed daily during the first week of life and weekly thereafter. Food intake in normal cages was measured at the age of 5, 6, 11, 13, 20, and 25 wk.
Metabolic studies including standardized analyses of glucose and lipid metabolism were performed at the age of 7, 15, and 30 wk. The following protocol was carried out at each time point: First, the animals were housed individually in a metabolic cage for 24 h allowing exact quantification of food and water intake as well as urine excretion. Next, they were fasted overnight, and a glucose tolerance test was performed by injecting ip glucose solution (2 mg glucose/g body weight) and measuring the glucose concentration in venous blood (obtained from the tail vein) using a Glucometer Elite XL device (Bayer, Leverkusen, Germany) before as well as 30, 60, 90, and 120 min after the injection. Two days later, the animals were again fasted overnight, and a retroorbital blood sample of 2 ml was collected during ether anesthesia within 30–60 sec. One of the six LIG rats died because of an anesthetic accident at wk 15. Blood analysis included the following parameters: fasted glucose (Glucometer), glycosylated hemoglobin (HbA1c, marker of the long-term course of circulating blood glucose) determined by standard procedures, rat insulin by ELISA (DRG Diagnostics, Marburg, Germany), rat leptin by ELISA (Phoenix Pharmaceuticals, Belmont, CA), rat visfatin by ELISA (Axxora, Lörrach, Germany), corticosterone and 11-dehydrocorticosterone by tandem mass spectrometry, total cholesterol, high-density lipoprotein (HDL) cholesterol (percentage of total cholesterol was calculated), triglycerides, and creatinine, the latter four parameters determined by standard procedures in a clinical routine laboratory. Urine creatinine was assayed, and creatinine clearance was calculated by standard procedures.
The animals were killed at an age of 30 wk under isoflurane anesthesia subsequent to fasting overnight. Epididymal fat tissue was carefully dissected bilaterally, weighed, and shock-frozen in liquid nitrogen. The central mesenteric fat pad at the division of the superior mesenteric artery superior into its branches was also dissected and weighed. Then, random samples of mesenteric fat, left rectus muscle, and right liver lobe were frozen in liquid nitrogen. All samples were stored at –70 C.
Quantitative RT-PCR
Leptin and visfatin mRNA in all frozen tissue samples was quantified by RT-PCR. One microgram of RNA was reverse-transcribed in the presence of an oligo-dT primer. The resulting cDNA was used as template for the amplification of the mRNA of interest using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) and the primers 5'-ATGACACCAAAACCCTCATCAAG-3' (leptin, forward), 5'-TGAAGTCCAAACCGGTGACC-3' (leptin, reverse), 5'-ATTCAAGGGGACGGAGTGGA-3' (visfatin, forward), and 5'-CTGTAGCAAAGCGCCACCAG-3' (visfatin, reverse). Real-time RT-PCR was performed on an iCycler iQ optical system (Bio-Rad). For each primer pair, amplicon size and reaction specificity were confirmed by agarose gel electrophoresis. Relative expression ratios were calculated by using the 
Ct method with a real-time PCR efficiency correction and both the porphobilinogen deaminase (PBGD) gene and the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene as references.
Statistical analysis
All data showed normal distribution. The data were compared using Kruskal-Wallis test with Bonferroni-adjusted Mann-Whitney U test used as a post test for bivariate comparison of single groups, two-way ANOVA with Bonferroni post tests, or Pearson correlation where appropriate. A P value (two-tailed) of <0.05 was considered to be statistically significant. All data are presented as mean ± SD.
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Results |
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. With respect to body weight, LIG rats caught up to SOP animals after 1 wk of life and to group C after 12 wk. The average body weight of the SOP group also reached that of group C at the age of 12 wk. After catch-up growth, parallel weight gain of the three groups was observed until the end of this study.
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Quantification of the epididymal fat at the age of 30 wk revealed a significantly increased fat pad mass in LIG animals compared both with SOP (P = 0.001) and C (P < 0.001). Interestingly, in SOP rats, the weight of the epididymal fat was also significantly higher than in group C (P < 0.001). Similarly, the amount of the mesenteric fat pad was also clearly increased in LIG animals compared with SOP and group C and in SOP rats compared with group C (Table 1
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Postnatal glucose metabolism
Next, we examined the glucose metabolism in the three experimental groups by measuring fasted plasma glucose, HbA1c, and insulin (Table 2) and performing standardized glucose tolerance tests. Fasted plasma glucose concentrations were similar in all groups at the age of 7, 15, and 30 wk except for an elevation in the LIG group compared with SOP animals at the age of 7 wk (P = 0.017). Glucose tolerance was impaired in the LIG group compared with SOP at all time points with significant results at 15 and 30 wk as well as in comparison with group C at 7 and 15 wk (Fig. 1, A–C). Moreover, the LIG group showed significantly elevated time-integrated glucose levels at 15 and 30 wk compared with SOP and at 7 and 15 wk compared with group C (Fig. 1D). In the SOP group, glucose tolerance was similar to that of group C at 7 and 15 wk (Fig. 1, A and B) and tended to be even better at 30 wk (Fig. 1C). SOP animals showed significantly lower time-integrated glucose concentration than groups LIG and C at wk 30 (Fig. 1D).
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In LIG animals, fasted serum insulin was reduced at the age of 7 wk (LIG vs. SOP, P = 0.002; LIG vs. C, P = 0.003), similar to that of groups SOP and C at the age of 15 wk, and clearly elevated at the age of 30 wk (LIG vs. SOP, P = 0.055; LIG vs. C, P = 0.004). The respective differences between SOP and group C were not significant (Table 2).
Glucocorticoids
Corticosterone plasma concentration was elevated in the LIG group compared with both SOP and group C at all time points (Table 2) with significant results at 15 wk (LIG vs. SOP, P = 0.013; LIG vs. C, P = 0.017) and 30 wk (LIG vs. SOP, P = 0.009; LIG vs. C, P = 0.017). SOP and group C animals showed similar corticosterone levels. The ratio of 11-dehydrocorticosterone to corticosterone (Table 2), calculated to estimate 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity, was similar in all groups except for a significantly decreased activity in LIG animals compared with SOP at wk 15 (P = 0.007).
Plasma lipids, leptin, and visfatin
In LIG and SOP animals, fasted triglyceride concentrations in serum were similar but found to be elevated relative to group C at all time points investigated (Table 2) with significant results at 15 wk (LIG vs. C, P = 0.009; SOP vs. C, P = 0.013) and 30 wk (LIG vs. C, P = 0.004; SOP vs. C, P = 0.017). The differences between LIG/SOP and group C rats increased with age. Total cholesterol of the overnight-fasted rats showed a comparable profile (Table 2) with significant results at 7 wk (SOP vs. C, P = 0.028) and 15 wk (LIG vs. C, P = 0.017; SOP vs. C, P = 0.017). Fasted HDL cholesterol concentrations (percentage of total plasma cholesterol) were similar in LIG and SOP animals at 7 and 15 wk of age but showed a relative decline in the LIG group at the age of 30 wk (LIG vs. SOP, P = 0.024). In LIG rats, HDL cholesterol levels were lower than in group C at all time points investigated (Table 2; 7 wk, P = 0.009; 15 wk, P = 0.057; 30 wk, P = 0.017). In SOP animals, HDL cholesterol showed the same tendency and was revealed to be significantly decreased in comparison with group C at 15 wk of age (P = 0.010).
Basal serum leptin concentration was comparable in all experimental groups at the age of 7 wk but clearly elevated in the LIG rats at the age of 15 wk (LIG vs. SOP, P = 0.007; LIG vs. C, P = 0.004) and 30 wk (LIG vs. SOP, P = 0.013; LIG vs. C, P = 0.004; Table 2). In SOP animals, basal leptin concentration was also increased in comparison with group C at the age of 30 wk (P = 0.009). The leptin concentrations at this last time point correlated strongly with the epididymal fat mass (Fig. 2) and the central mesenteric fat pad mass (r2 = 0.45; P < 0.001). Fasted serum visfatin concentrations did not differ significantly among the three groups at any time (Table 2).
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Impact of IUGR on leptin and visfatin expression
After killing of all animals at 30 wk of age, leptin and visfatin mRNA in liver, muscle, epididymal fat, and mesenteric fat samples were quantified by real-time PCR. All data presented in the text and Fig. 3 are normalized to the housekeeping gene PBGD. Leptin expression was similar in LIG and SOP as well as group C animals in all tissues investigated. Visfatin expression also did not differ significantly between the three experimental groups in both epididymal fat (visfatin/PBGD in C, 1.03 ± 0.14 relative units) and mesenteric fat (visfatin/PBGD in C, 1.01 ± 0.17 relative units). However, visfatin expression in mesenteric fat correlated positively with plasma visfatin (r2 = 0.36; P = 0.004). In muscle tissue, the relative amounts of visfatin mRNA in LIG and SOP rats were similar but reduced in comparison with the animals of group C (Fig. 3A). One outlier (by Grubbs test) was excluded from further analysis. Visfatin expression in the muscle correlated inversely with fasted serum insulin (r2 = 0.34; P = 0.007) but not with circulating glucose or visfatin or with epididymal or mesenteric fat pad mass. In liver tissue, visfatin expression in the samples from LIG and SOP animals did not differ significantly but was also found to be decreased relative to group C (Fig. 3B). Visfatin expression in the liver correlated inversely with epididymal fat mass (r2 = 0.29; P = 0.008) but did not show a significant correlation with fasted plasma insulin, glucose, or visfatin concentrations or with mesenteric fat pad mass. Normalization of leptin and visfatin expression to another housekeeping gene, HPRT, yielded similar results.
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Discussion |
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Comparable to our findings concerning glucose metabolism, we observed an elevation of circulating corticosterone concentration only in the offspring of ligated dams at all time points investigated. In adult men, fasting plasma cortisol levels are inversely related to birth weight and positively associated with both impaired glucose tolerance and elevated circulating triglycerides (20). Therefore, the increased glucocorticoid levels may explain the disturbed glucose tolerance and lipid metabolism in LIG offspring. Children with low 11β-HSD2 activity show impaired catch-up growth, high lipid levels, and insulin resistance (21). However, our LIG animals revealed fast catch-up growth and no insulin resistance at the age of 7 wk. Furthermore, the hypothesis that a reduced 11β-HSD2 activity contributes to increased plasma corticosterone and disturbed glucose metabolism in IUGR offspring is not clearly supported by our data, because LIG animals show increased corticosterone levels at normal 11β-HSD2 activity at wk 7 and 30. This could be explained by elevated endogenous corticosterone production in our LIG animals, possibly due to increased stress sensitivity.
Most interestingly, however, long-lasting changes in lipid metabolism leading to an increase in the serum concentrations of triglycerides, cholesterol, and leptin as well as an elevation of body fat content in our animals were not only induced by uterine artery ligation but also by sham operation. This phenomenon has not yet been reported. It may be explained either by postaggression metabolism, which is typically associated with elevated glucocorticoid levels as well as a reduced utilization of glucose and fat (22) of the dam or by reduced nutrient intake of the pregnant animal for several hours after the operation, which may suffice to program the lipid metabolism toward increased body fat content and leptin resistance. In the offspring, a possible explanation for the altered lipid metabolism could be provided by increased glucocorticoid levels. However, we show that elevation of lipids in adult SOP animals is not associated with increased corticosterone levels or with decreased 11β-HSD2 activity.
In the existing literature, no catch-up growth of LIG compared with SOP offspring has been described for Wistar rats (11), whereas catch-up growth within the first week of life has been observed in Sprague Dawley rats (10), both after litter size reduction to four to eight pups. Our study, which provides the first data on food intake in this animal model, indicates a parallel weight gain after initial catch-up growth (LIG vs. SOP). Interestingly, the catch-up growth of the LIG animals occurred under relatively low food consumption, indicating reduced energy expenditure in the LIG group. In contrast, catch-up of SOP rats to group C required significantly higher food intake until the age of 7 wk. However, plasma levels of leptin, a potential regulator of food intake and energy expenditure (23), were similar in all groups at 7 wk of age, confirming data of a previous study (11). Therefore, we conclude that the mechanisms underlying body weight catch-up are independent of circulating leptin and may actually differ between LIG and SOP animals. At 30 wk of age, however, examination of the epididymal and mesenteric fat pad masses as indicators of body fat content (24, 25) as well as serum leptin revealed that, despite similar body weights, not only LIG compared with SOP (10) but also SOP compared with group C animals showed an altered body composition with higher fat content. Leptin levels correlated strongly with body fat mass. Furthermore, higher food intake and concomitantly elevated serum leptin concentrations in LIG and partially also in SOP compared with group C animals indicated leptin resistance not only in the LIG rats but to a certain extent also in the SOP animals at this point of time.
Both LIG and SOP animals developed hypertriglyceridemia, and pathological changes were also seen with respect to circulating total cholesterol. This may be explained by a stimulation of lipolysis or by decreased adipogenesis (26). Although both may be due to a reduced efficacy of insulin (26), LIG animals show decreased serum insulin concentrations at the age of 7 wk and increased insulin levels at 30 wk of age, indicating insulin resistance, whereas no corresponding changes were observed in SOP animals. Because in both groups, visfatin expression in muscle and liver was reduced, this adipocytokine may provide an alternative explanation for the increased triglyceride and cholesterol levels associated with a poor HDL/total cholesterol ratio, which indicate a programmed disorder of lipid metabolism likely to be involved in the known pathological sequelae of IUGR. However, serum visfatin concentrations did not differ between the groups and correlated with visfatin expression only in mesenteric fat tissue. Therefore, our experimental data do not support the recently published finding of an inverse association between plasma visfatin and circulating triglycerides and a positive correlation with HDL cholesterol in humans (27). Moreover, considering the reported insulin-mimetic effects of visfatin in organs of glucose homeostasis (15), one would expect impaired glucose tolerance in LIG and SOP rats, which was observed only in LIG animals. Visfatin expression in the liver showed no correlation with fasted plasma glucose or insulin concentrations. Thus, visfatin does not appear to be implicated in the disturbed glucose metabolism and is likely to represent only a marker of fat accumulation.
Additionally, an interesting observation of our study is that not only offspring of dams treated by uterine artery ligation but also offspring of sham-operated animals show an elevated creatinine clearance at the age of 7 and 15 wk, suggesting glomerular hyperfiltration. IUGR leads to a reduced number of glomeruli in humans (28), which may predispose to the development of hyperfiltration followed by glomerulosclerosis and hypertension in later life (29). In rats, programming of hypertension and kidney disease has been induced almost exclusively by malnutrition (30) or glucocorticoid treatment (31), whereas similar reports regarding the model of uterine artery ligation are rare (32). This may be due to the fact that LIG and SOP pups are similar with respect to renal function at least until the age of 30 wk as shown here.
In conclusion, this study on an established rat model of IUGR has elucidated that uterine artery ligation but not sham operation of the dam may result in developmental programming of an impaired glucose tolerance and elevated glucocorticoid levels in the offspring, whereas pathogenic programming of lipid metabolism, body composition, and renal pathology can be induced both by uterine artery ligation and by sham operation. Thus, the use of this animal model of IUGR requires both SOP and untreated controls. Furthermore, our study indicates that visfatin is not involved in the pathogenesis of the disturbed glucose metabolism subsequent to IUGR and may represent only a marker of fat accumulation.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online December 6, 2007
Abbreviations: HbA1c, Glycosylated hemoglobin; HDL, high-density lipoprotein; 11β-HSD2, 11β-hydroxysteroid dehydrogenase type 2; IUGR, intrauterine growth restriction; LIG, bilateral uterine artery ligation; pc, postcoitum; SOP, sham operated.
Received July 3, 2007.
Accepted for publication November 26, 2007.
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, P < 0.05; 
