Uteroplacental Insufficiency after Bilateral Uterine Artery Ligation in ...

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Endocrinology 149(3):1056 –1063 Copyright © 2008 by The Endocrine Society doi: 10.1210/en.2007-0891

Uteroplacental Insufficiency after Bilateral Uterine Artery Ligation in the Rat: Impact on Postnatal Glucose and Lipid Metabolism and Evidence for Metabolic Programming of the Offspring by Sham Operation Kai-Dietrich Nu¨sken, Jo¨rg Do¨tsch, Manfred Rauh, Wolfgang Rascher, and Holm Schneider 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 Ligation of the uterine arteries (LIG) in rats serves as a model of intrauterine growth restriction and subsequent developmental programming of impaired glucose tolerance, hyperinsulinemia, and adiposity in the offspring. Its impact on lipid metabolism has been less well investigated. We compared parameters of glucose and lipid metabolism and glucocorticoid levels in the offspring of dams that underwent either LIG or sham operation (SOP) with those of untreated controls. Blood parameters including insulin, leptin, and visfatin as well as body weight, food intake, and creatinine clearance were recorded up to an age of 30 wk. Glucose tolerance tests were performed, and both leptin and visfatin expression in liver, muscle, and epididymal and mesenteric fat was quantified by RT-PCR. After catch-up growth, weight gain of all groups was similar, despite lower food intake of the LIG rats. LIG off-

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N INDUSTRIALIZED COUNTRIES, placental insufficiency is the most important reason for intrauterine growth restriction (IUGR) (1), which may predispose the fetus to the development of adiposity, diabetes mellitus type 2, and cardiovascular diseases in later life (2), depending on early postnatal nutrition (3). To investigate potential sequelae of IUGR, ligation of the uterine arteries in rats has been used frequently as an animal model of uteroplacental insufficiency. Offspring of dams that underwent bilateral uterine artery ligation (LIG), compared with offspring of sham-operated animals (SOP), show reduced body length and weight at birth together with hypoglycemia (4, 5), hypoinsulinemia (5), and elevated concentrations of circulating corticosterone (6). In the affected neonates, the levels of circulating triglycerides (7, 8) and leptin (9) are unaltered. In adulthood, LIG animals are characterized by hyperglycemia, insulin resistance progressing to type 2 diabetes, obesity (10), and hypertriglyceridemia (8). The serum leptin concentra-

First Published Online December 6, 2007 Abbreviations: HbA1c, Glycosylated hemoglobin; HDL, high-density lipoprotein; 11␤-HSD2, 11␤-hydroxysteroid dehydrogenase type 2; IUGR, intrauterine growth restriction; LIG, bilateral uterine artery ligation; pc, postcoitum; SOP, sham operated. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

spring showed impaired glucose tolerance from the age of 15 wk as well as elevated glycosylated hemoglobin and corticosterone levels. However, the body fat content of both LIG and SOP animals increased relative to controls, and both showed elevated triglyceride, total cholesterol, and leptin levels as well as a reduced proportion of high-density lipoprotein cholesterol. Thus, use of the LIG model requires both SOP and untreated controls. Although only LIG is associated with impaired glucose tolerance, pathogenic programming of the lipid metabolism can also be induced by SOP. Visfatin does not appear to be involved in the disturbed glucose metabolism after intrauterine growth restriction and may represent only a marker of fat accumulation. (Endocrinology 149: 1056 –1063, 2008)

tion is similar to that of SOP animals (11). Data on circulating cholesterol have not been reported for LIG or for SOP rats. 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

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Nu¨sken et al. • Metabolic Programming in the Rat

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. Materials and Methods Surgical procedures and selection of the pups All procedures on animals were conducted in accordance with the German regulations and legal requirements. The experimental protocol was approved by the appropriate Institutional and Governmental Review Boards. 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-

Endocrinology, March 2008, 149(3):1056 –1063

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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, Lo¨rrach, Germany), corticosterone and 11-dehydrocorticosterone by tandem mass spectrometry, total cholesterol, highdensity 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.

Results Growth, food intake, and body fat content of the animals

The gain of body weight and the course of absolute and weight-adjusted daily food intake of LIG, SOP, and control animals (group C) are summarized in Table 1. 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. Average food intake was significantly lower in LIG rats compared with SOP animals at almost all time points until wk 20, that is wk 5 (P ⫽ 0.010), wk 6 (P ⫽ 0.010), wk 7 (P ⫽ 0.017), wk 11 (P ⫽ 0.017), wk 15 (P ⫽ 0.002), and wk 20 (P ⫽ 0.002). LIG rats also showed significantly lower food intake when compared with group C at the age of 5, 7, 11, 13, and 20 wk, whereas the food intake of SOP rats was significantly lower than that of group C only at the age of 11 (P ⬍ 0.001), 15 (P ⬍ 0.001), and 20 (P ⬍ 0.001) weeks. At 25 wk of age, however, food intake of LIG rats was significantly increased in comparison with group C (P ⬍ 0.001). Quantification of the epididymal fat at the age of 30 wk

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TABLE 1. Postnatal weight gain and nutritional parameters in the IUGR model Age (wk)

Body weight (g)

Food intake (g) Food intake/body weight (g/kg) Epididymal fat mass (g) Central mesenteric fat pad (g)

0 1 7 15 30 7 15 30 7 15 30 30 30

Experimental groups LIG (n ⫽ 6)

SOP (n ⫽ 12)

C (n ⫽ 6)

P value (Kruskal-Wallis)

4.77 ⫾ 0.34 11.1 ⫾ 0.9 249 ⫾ 10 429 ⫾ 13 521 ⫾ 26 22.7 ⫾ 3.4 25.1 ⫾ 2.7 26.8 ⫾ 2.9 80.9 ⫾ 10.2 57.8 ⫾ 5.9 51.4 ⫾ 3.8 4.95 ⫾ 1.29 0.33 ⫾ 0.03

6.13 ⫾ 0.18 11.7 ⫾ 1.3 257 ⫾ 24 418 ⫾ 39 513 ⫾ 53 26.9 ⫾ 2.5 26.8 ⫾ 2.4 26.5 ⫾ 4.8 94.5 ⫾ 10.5 63.3 ⫾ 6.6 52.7 ⫾ 13.8 3.05 ⫾ 1.17 0.20 ⫾ 0.04

6.85 ⫾ 0.26 14.4 ⫾ 0.3 291 ⫾ 12 448 ⫾ 21 529 ⫾ 36 26.3 ⫾ 2.3 30.7 ⫾ 2.4 22.0 ⫾ 4.0 82.5 ⫾ 8.3 67.7 ⫾ 3.7 41.6 ⫾ 7.3 1.88 ⫾ 0.20 0.14 ⫾ 0.02

⬍0.001 0.001 0.005 NS NS 0.031 0.005 NS 0.023 0.012 NS ⬍0.001 ⬍0.001

C, Offspring of untreated control dams; LIG, offspring of dams that underwent bilateral uterine artery ligation; SOP, offspring of shamoperated animals. P value of Kruskal-Wallis test (LIG, SOP, and C) is shown. NS, Not significant.

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).

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 ani-

TABLE 2. Course of key parameters of glucose and lipid metabolism in the IUGR model Age (wk)

Glucose (fasted, mg/dl) HbA1c (%) Insulin (fasted, ng/ml) Leptin (fasted, pg/ml) Visfatin (fasted, ng/ml) Corticosterone (fasted, ng/ml) 11-Dehydrocorticosterone/corticosterone (fasted, ng/100 ng) Triglycerides (fasted, mg/dl) Total cholesterol (fasted, mg/dl) HDL cholesterol (fasted, % of total cholesterol)

Experimental groups LIG (n ⫽ 6)

SOP (n ⫽ 12)

C (n ⫽ 6)

P value (Kruskal-Wallis)

7 15 30 7 15 30 7 15 30 7 15 30 7 15 30 7 15 30 7

91.5 ⫾ 8.9 78.5 ⫾ 7.6 62.4 ⫾ 7.2 3.91 ⫾ 0.05 4.59 ⫾ 0.14 4.05 ⫾ 0.12 0.10 ⫾ 0.04 1.03 ⫾ 0.37 0.91 ⫾ 0.12 476 ⫾ 104 1381 ⫾ 228 2188 ⫾ 1048 0.23 ⫾ 0.22 0.44 ⫾ 0.48 0.06 ⫾ 0.08 504 ⫾ 189 550 ⫾ 193 527 ⫾ 225 4.83 ⫾ 1.34

79.2 ⫾ 7.9 77.2 ⫾ 8.7 68.2 ⫾ 8.4 3.92 ⫾ 0.10 4.31 ⫾ 0.20 3.92 ⫾ 0.12 0.47 ⫾ 0.43 1.20 ⫾ 0.76 0.46 ⫾ 0.39 689 ⫾ 585 879 ⫾ 396 968 ⫾ 532 0.02 ⫾ 0.03 0.29 ⫾ 0.29 0.41 ⫾ 0.54 322 ⫾ 154 238 ⫾ 151 157 ⫾ 107 6.20 ⫾ 2.28

86.3 ⫾ 14.9 76.7 ⫾ 8.6 68.0 ⫾ 7.6 3.94 ⫾ 0.07 4.23 ⫾ 0.08 3.85 ⫾ 0.14 0.76 ⫾ 0.28 0.83 ⫾ 0.48 0.31 ⫾ 0.10 417 ⫾ 143 558 ⫾ 155 452 ⫾ 10 0.12 ⫾ 0.18 0.38 ⫾ 0.35 0.04 ⫾ 0.07 303 ⫾ 236 218 ⫾ 95 171 ⫾ 134 3.80 ⫾ 1.70

0.076 NS NS NS 0.015 NS 0.001 NS 0.024 NS 0.004 0.001 NS NS NS NS 0.021 0.017 NS

15 30 7 15 30 7 15 30 7 15 30

3.11 ⫾ 0.43 4.27 ⫾ 2.03 53.8 ⫾ 3.6 88.3 ⫾ 17.7 132.2 ⫾ 30.6 77.6 ⫾ 12.2 81.7 ⫾ 10.0 118.0 ⫾ 25.2 84.2 ⫾ 4.3 88.1 ⫾ 1.7 80.8 ⫾ 4.3

4.70 ⫾ 1.24 6.15 ⫾ 2.08 48.4 ⫾ 19.4 83.4 ⫾ 26.7 114.2 ⫾ 49.5 81.6 ⫾ 12.1 85.3 ⫾ 20.9 124.9 ⫾ 48.2 86.9 ⫾ 3.9 87.0 ⫾ 2.4 86.6 ⫾ 4.5

3.99 ⫾ 1.33 4.86 ⫾ 1.34 28.9 ⫾ 7.3 45.6 ⫾ 16.1 59.8 ⫾ 22.0 67.3 ⫾ 9.9 60.3 ⫾ 11.6 82.3 ⫾ 12.9 90.8 ⫾ 3.6 90.6 ⫾ 2.2 89.4 ⫾ 4.3

0.015 NS NS 0.014 0.007 0.073 0.024 NS 0.034 0.018 0.022

C, Offspring of untreated control dams; LIG, offspring of dams that underwent bilateral uterine artery ligation; SOP, offspring of shamoperated animals. P value of Kruskal-Wallis test (LIG, SOP, and C) is shown. NS, Not significant.


mals 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). These results were in agreement with the HbA1c levels, which were similar in all groups at the age of 7 wk but significantly increased in LIG rats as compared with SOP animals at the age of 15 wk (P ⫽ 0.013) or with group C at


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the age of 15 (P ⫽ 0.004) as well as 30 wk (P ⫽ 0.052). There were no significant differences of the HbA1c levels between groups SOP and C at the three time points investigated. 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 ⫽

FIG. 1. Postnatal glucose tolerance in IUGR rats. Glucose tolerance tests were performed at the age of 7 wk (A), 15 wk (B), and 30 wk (C); D, time-integrated glucose levels during glucose tolerance tests at the age of 7, 15, and 30 wk. All data are presented as mean ⫾ SD. C, Offspring of untreated control dams; LIG, offspring of dams that underwent bilateral uterine artery ligation; SOP, offspring of sham-operated animals. LIG compared with SOP: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. LIG compared with C: †, P ⬍ 0.05; ††, P ⬍ 0.01; †††, P ⬍ 0.001. SOP compared with C: #, P ⬍ 0.05; ##, P ⬍ 0.01; ###, P ⬍ 0.001.

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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 11␤-hydroxysteroid dehydrogenase type 2 (11␤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). Creatinine clearance

Creatinine clearance (milliliters per kilogram per minute) was calculated to estimate the renal function. In comparison with group C (5.10 ⫾ 0.38), LIG (8.61 ⫾ 1.12; P ⫽ 0.002) and SOP rats (9.91 ⫾ 1.22; P ⬍ 0.001) showed hyperfiltration with increased creatinine clearance at 7 wk. The same was found in LIG (10.08 ⫾ 1.89) and SOP animals (8.97 ⫾ 1.50) compared with group C (7.46 ⫾ 0.83) at 15 wk (LIG vs. C, P ⫽ 0.017; SOP vs. C, P ⫽ 0.044). At 30 wk, all three groups revealed a similar renal function (LIG, 5.59 ⫾ 0.58; SOP, 5.80 ⫾ 1.40; C, 6.01 ⫾ 0.90). 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. Discussion

FIG. 2. Correlation of serum leptin concentration with the epididymal fat mass. Correlation coefficient and P value are indicated.

In this study on an established but not uncontroversial animal model of IUGR (10, 12, 17), we confirmed that uterine artery ligation of the dam results in developmental programming of impaired glucose tolerance and subsequent hyperinsulinemia in the offspring. Disturbed glucose metabolism was observed only in animals affected by uterine artery ligation but not in the offspring of sham-operated animals. Signs of insulin resistance were evident as described previously (5, 10), but the diagnosis was confirmed by increased levels of HbA1c, which is determined by insulin secretion and sensitivity (18). Thus, our data from Wistar rats are in agreement with those from Sprague Dawley rats (10). How-


FIG. 3. Impact of IUGR on visfatin expression. Visfatin mRNA in muscle (A) and liver (B) was quantified by real-time PCR. Gene expression data normalized to the housekeeping gene PBGD are shown. C, Offspring of untreated control dams; LIG, offspring of dams that underwent bilateral uterine artery ligature; SOP, offspring of shamoperated animals. P values obtained by Mann-Whitney U test are indicated.

ever, fasted plasma glucose concentrations did not differ among the three experimental groups at the time points investigated, and our animals did not develop type 2 diabetes within the time frame of this study. Furthermore, our data do not support the conclusion of a recent study by Pfab et al. (19) that elevated blood glucose concentrations and increased HbA1c levels in individuals affected by IUGR may be present as early as in utero, because in our animal model, an inverse correlation between birth weight and HbA1c was not detectable before the age of 15 wk. However, this exciting issue certainly deserves further investigation. 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


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glucocorticoid levels may explain the disturbed glucose tolerance and lipid metabolism in LIG offspring. Children with low 11␤-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 11␤-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 11␤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 11␤-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.

1062 Endocrinology, March 2008, 149(3):1056 –1063

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. Acknowledgments We thank Yvonne Birkner and Elisabeth Koppmann for animal care and technical assistance and the staff of our clinical laboratory for their help with measurements.


Received July 3, 2007. Accepted November 26, 2007. Address all correspondence and requests for reprints to: Kai-Dietrich Nu¨sken, M.D., Department of Pediatrics, Friedrich-Alexander-University Erlangen-Nuernberg, Loschgestrasse 15, 91054 Erlangen, Germany. E-mail: [email protected]. This work was supported by the “Erlanger Leistungsbezogene Anschubfinanzierung und Nachwuchsfo¨rderung” of the University of Erlangen-Nuernberg (ELAN, Grant AZ04.10.26.1; to K.-D.N.) and the German Research Foundation (DFG, Grant D0682/33; to J.D.). Disclosure Statement: The authors have nothing to disclose.

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