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Endocrinology Vol. 142, No. 9 3964-3973
Copyright © 2001 by The Endocrine Society

ARTICLES

IGF-I Treatment Reduces Hyperphagia, Obesity, and Hypertension in Metabolic Disorders Induced by Fetal Programming

Mark H. Vickers, Bettina A. Ikenasio and Bernhard H. Breier

Liggins Institute for Medical Research, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand 92019

Address all correspondence and requests for reprints to: Associate Professor Bernhard H. Breier, Liggins Institute for Medical Research, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: bh.breier{at}auckland.ac.nz


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The discovery of a link between in utero experience and later metabolic and cardiovascular disease is one of the most important advances in epidemiology research of recent years. There is increasing evidence that alterations in the fetal environment may have long-term consequences on cardiovascular, metabolic, and endocrine pathophysiology in adult life. This process has been termed programming, and we have shown that undernutrition of the mother during gestation leads to programming of hyperphagia, obesity, hypertension, hyperinsulinemia, and hyperleptinemia in the offspring. Using this model of maternal undernutrition throughout pregnancy combined with postnatal hypercaloric nutrition of the offspring, we examined the effects of IGF-I therapy. Virgin Wistar rats (age 75 ± 5 d, n = 20 per group) were time mated and randomly assigned to receive food either ad libitum or 30% of ad libitum intake (UN) throughout pregnancy. At weaning, female offspring were assigned to one of two diets (control or hypercaloric [30% fat]). Systolic blood pressure was measured at day 175 and following infusion with 3 µg/g per day recombinant human IGF-1 (rh-IGF-I) by minipump for 14 d. Before treatment, UN offspring were hyperinsulinemic, hyperleptinemic, hyperphagic, obese, and hypertensive on both diets, compared with ad libitum offspring and this was exacerbated by hypercaloric nutrition. IGF-I treatment increased body weight in all treated animals. However, systolic blood pressure, food intake, retroperitoneal and gonadal fat pad weights, and plasma leptin and insulin concentrations were markedly reduced with IGF-I treatment. IGF-I treatment resulted in a 3- to 5-fold increase in 38–44 kDa and 28–30 kDa IGF binding proteins, although in UN animals, there was an impaired and differential up-regulation of these insulin-like growth factor binding proteins following IGF-I treatment. The 24-kDa IGF binding protein representing IGF binding protein-4 was down-regulated in all IGF-I-treated animals, but the decrease was more marked in UN animals. Our data suggest that IGF-I treatment alleviates hyperphagia, obesity, hyperinsulinemia, hyperleptinemia, and hypertension in rats programmed to develop the metabolic syndrome X.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS INCREASING evidence that metabolic disorders that manifest in adult life have their roots before birth. This concept of fetal programming is based on epidemiological and experimental observations of close associations between an adverse intrauterine environment and the later onset of adult metabolic and cardiovascular disorders (1, 2, 3). We have defined fetal programming as an adaptive process to an adverse intrauterine environment that alters the fetal metabolic and hormonal milieu, resulting in resetting of developmental processes to ensure fetal survival. The persistence of these adaptive responses, designed for survival in a fetal environment, into postnatal life, leads to metabolic and cardiovascular disorders (4).

We have developed an animal model of fetal programming in which we apply maternal undernutrition throughout gestation, generating a nutrient-deprived intrauterine environment that results in fetal growth retardation and postnatal growth failure and leads to changes in allometric growth patterns and endocrine parameters of the somatotrophic axis (5, 6). We have recently shown that programmed offspring develop profound hyperphagia, obesity, hypertension, hyperinsulinemia, and hyperleptinemia during adult life and that postnatal hypercaloric nutrition amplifies the metabolic and cardiovascular abnormalities induced by fetal programming (4). Thus, our animal model closely resembles the clinical and metabolic abnormalities seen in humans born of low birth weight and, furthermore, displays the phenotype of syndrome X (7, 8). Epidemiological studies have shown that babies born of low birth weight develop increased rates of obesity in adult life (9). This was most clearly shown in a recent report from the Dutch Famine Study in which poor nutrition in the first trimester of pregnancy resulted in increased rates of obesity during adult life (10). Animal studies have also shown that maternal malnutrition during pregnancy results in the development of adult-onset obesity in offspring (9, 11, 12).

IGF-I is one of the most important regulators of growth, and IGF-I deficiency is associated with prenatal and postnatal growth failure (13, 14). Under conditions of adequate nutrition, IGF-I has been shown to promote postnatal catch-up growth in rats with intrauterine growth retardation caused by gestational protein deficiency (15). IGF-I therapy is associated with increased insulin sensitivity in normal subjects as well as in patients with GH deficiency, type 2 diabetes mellitus, and type A insulin resistance (16). IGF-I can reduce hyperglycemia in patients with severe insulin resistance by direct effects mediated via the IGF-I receptor (17). IGF-I infusion lowers insulin and lipid levels in healthy humans and reduces plasma leptin concentrations in rats (18), suggesting that IGF-I may reduce the degree of insulin resistance in type 2 diabetes, obesity, and hyperlipidemia (19). Clinical studies of IGF-I in hypertension are limited, but IGF-I has previously been shown to have vasodilatory effects and to improve cardiac function in healthy volunteers (20). In animal studies, IGF-I treatment has been shown to cause partial reversion of hypertension-induced changes in cardiac function and to increase cardiac output and stroke volume (21). Furthermore, recent evidence suggests that IGF-I can interact with the renin-angiotensin system (RAS) and may alter angiotensin II expression via angiotensin type 1 receptor regulation (22, 23). The reported effects of IGF-I on cardiovascular and metabolic homeostasis may be mediated by the IGF-binding proteins (IGFBPs). IGFBP-1 and -2 levels closely reflect changes related to nutrition, insulin secretion, and disease states such as obesity and type 2 diabetes. IGFBP-3 correlates with IGF-I and is a chronic indicator of GH- dependent growth status (24). Previous work by our group (5) and others (25, 26) has shown differential expression of IGFBPs following fetal growth retardation.

The present study investigates the morphometric, metabolic, and endocrine responses to IGF-I treatment in postnatal life following fetal programming alone or in combination with hypercaloric nutrition. The aim of the present study was to establish whether IGF-I treatment can alleviate hyperinsulinemia, hyperleptinemia, hyperphagia, obesity, and hypertension caused by fetal programming and postnatal hypercaloric nutrition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Virgin Wistar rats (age 100 ± 5 d, n = 15 per group) were time mated using a rat oestrus cycle monitor to assess the stage of oestrus of the animals before introducing the male. After confirmation of mating, rats were housed individually in standard rat cages containing wood shavings as bedding and free access to water. All rats were kept in the same room with a constant temperature maintained at 25 C and a 12-h light:12-h darkness cycle. Animals were assigned to one of two nutritional groups: group 1 consisted of undernutrition ([UN], 30% of ad libitum) of a standard diet throughout gestation; group 2 consisted of a standard diet fed ad libitum (AD) throughout pregnancy. Food intake and maternal weights were recorded daily until birth. After birth, pups were weighed and litter size recorded. Pups from undernourished mothers were cross-fostered onto dams that received AD feeding throughout pregnancy. Litter size was adjusted to eight pups per litter to assure adequate and standardized nutrition until weaning. After weaning, female offspring from the two groups of dams a) AD offspring and b) offspring from UN mothers were divided into two balanced postnatal nutritional groups to be fed either a standard diet (total digestible energy 2959 kcal/kg, protein 19.4%, fat 5%, fat/energy ratio 15.21%, protein energy ratio 26.23) or a hypercaloric diet (total digestible energy 4846 kcal/kg, protein 31.8%, fat 30%, fat/energy ratio 55.72%, protein/energy ratio 26.25%). The mineral and vitamin content in the two diets were identical and in accordance with the requirements for standard rat diets. The fat content of the hypercaloric diet is typical of that seen in many Western diets. Weights and food intake of all offspring were measured daily for the first 2 wk and then every second day. At day 175, systolic blood pressure measurements were recorded using tail cuff plethysmography. Rats were then weight matched and received either rh-IGF-I (3 µg/g per day) or saline by osmotic minipump (model 2002, Alzet Corp., Palo Alto, CA) for 14 d. On the day before the rats were killed, a repeated systolic blood pressure was recorded. Rats were then fasted overnight and killed by halothane anesthesia followed by decapitation. Blood was collected into heparinized vacutainers and stored on ice until centrifugation and removal of supernatant for analysis. All animal work was approved by the Animal Ethics Committee of the University of Auckland.

Blood pressure measurements
Systolic blood pressure (SBP) was recorded by tail cuff plethysmography according to the manufacturer’s instructions (blood pressure analyser IITC, Life Science, Woodland Hills, CA). Rats were restrained in a clear plastic tube in a prewarmed room (25–28 C). After the rats had acclimatized (10–15 min), the cuff was placed on the tail and inflated to 240 mm Hg. Pulses were recorded during deflation at a rate of 3 mm Hg/sec, and reappearance of a pulse was used to determine SBP. A minimum of three clear SBP recordings per animal was taken, and the coefficient of variation for repeated measurements was <5%.

IGF-I infusion
At day 175, rats were weight matched (n = 6 per group) and received either rh-IGF-I (Genentech, Inc., San Francisco, CA, code no. G117AZ, batch c9831AY) or saline by osmotic minipump (model 2002, Alzet Corp.). The dose was 3 µg/g per day for 14 days with a pump delivery rate of 5 µl/h. The mean pump rate for the batch (lot no. 167258) of pumps used was 5.23 ± 0.2 µl/h. Pumps containing the IGF-I or saline solution were incubated in sterile saline for 4 h at 37 C before implantation. The osmotic pumps were implanted sc, under halothane anesthesia, using a small incision made in the skin between the scapulae. Using a hemostat, a small pocket was formed by spreading apart the sc connective tissues. The pump was inserted into the pocket with the flow moderator pointing away from the incision. The skin incision was then closed with sutures. All animals (n = 48) were housed individually for the duration of the study.

Endocrine analyses
IGF-I in rat blood plasma was measured using an IGFBP-blocked RIA described previously (27). The ED50 was 0.1 ng/tube, and the intra- and interassay coefficients of variation were <5% and <10% respectively.

Rat insulin was measured by RIA as described previously (4). Blood plasma was diluted 1:4 in assay buffer (0.01 M PBS containing 0.37% Na EDTA and 0.5% BSA, pH 6.2). In brief, the primary antibody (guinea pig antiovine insulin) was diluted in assay buffer to an initial working dilution of 1:80,000. After 0.1 ml diluted sample, control, or standard (rat insulin, 0.01–10 ng/ml, Crystal Chem, Chicago, IL) was incubated with 0.2 ml primary antibody for 24 h at room temperature, 0.2 ml 125I-rh-Insulin (lot no. 615–707-208, Eli Lilly, Indianapolis, IN) was added at 15–20,000 counts per tube. Equilibrium conditions were established after 24-h incubation at 4 C. A second antibody was used to separate bound from free ligand as outlined previously (28) and the pellet counted by {gamma} counter. Rat plasma samples showed parallel displacement to the standard curve, and recovery of unlabeled rat insulin was 96.5 ± 4.4% (mean ± SEM, n = 11). The ED50 was 0.5 ng/ml.

A double-antibody RIA was developed for measurement of leptin in rat plasma. An antibody was raised in rabbits against a fragment (aa 30–45) of bovine leptin. Standard preparation was rm-leptin (Crystal Chem, #CR-6781) used in concentrations ranging from 0.5 to 20 ng/ml. Samples were assayed neat or diluted 1:2–1:4 in assay buffer (0.05 M PBS, pH 7.4 containing 0.1 M NaCl, 0.5% BSA, 10 mM EDTA, 0.05% NaN3). In brief, 100-µl primary antibody (1:25,000) was added to tubes containing 100-µl sample or standard. Following incubation for 24 h at 4 C, 100 µl tracer (125I-rm-leptin, 20 000 cpm per tube) was added to all tubes followed by a further incubation for 24 h at 4 C. A second antibody technique to separate bound from free ligand was used as outlined previously (28). Rat plasma samples showed parallel displacement to the standard curve, and recovery of unlabeled rm-leptin was 101.4 ± 2.7% (mean ± SEM, n = 26). The ED50 was 0.37 ng/ml, and the intra-assay coefficient of variation was < 5% (all samples measured within a single assay).

Fasting plasma glucose concentrations from samples taken at the time of sacrifice were measured using a glucose analyzer (model 2300, Yellow Springs Instrument Co., Yellow Springs, OH). Blood plasma FFA were measured by diagnostic kit (no. 1383175, Roche Molecular Biochemicals, Indianapolis, IN). All other plasma analytes were measured by a BM 737 analyzer (Hitachi, Roche Diagnostics, Indianapolis, IN) by Auckland Healthcare Laboratory Services.

IGFBPs in rat plasma (2-µl sample, n = 6 per treatment group) were analyzed by ligand blotting (29) as described in detail elsewhere (30). Rat 125I-IGF-II was used as radiolabel. Nitrocellulose blots were air dried and exposed to X-Omat AR diagnostic film (Eastman Kodak Co., Rochester, NY) in hyperscreen cassettes with intensifier screens (Amersham Pharmacia Biotech, Piscataway, NJ). For quantification, nitrocellulose blots were exposed overnight to phospor imaging screens and analyzed on a Storm PhosporImager system using ImageQuant software (Molecular Dynamics, Inc., Sky Valley, CA). All values were expressed relative to a normal rat plasma pool and standardized to 100% for control group. The IGFBPs were identified on the basis of their molecular size using nomenclature previously described (31).

Statistical analyses were carried out using SigmaStat (Jandel Scientific, San Rafael, CA) and StatView (SAS Institute, Inc., Cary, NC) statistical packages. Differences between groups were determined by two-way (pre-IGF-I treatment) or three-way ANOVA (post-IGF-I treatment) followed by Bonferroni post hoc analysis, and data are shown as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maternal undernutrition resulted in fetal growth retardation reflected by significantly decreased body weight at birth in the offspring from UN dams (UN 4.02 ± 0.03 g, AD 6.13 ± 0.04 g, P < 0.001). Litter size was not different between the two groups (AD 11.7 ± 1.93, UN 11.2 ± 2.03). From birth until weaning at day 22, body weights remained significantly lower in the UN offspring (AD 51.5 ± 0.6 g, UN 37.8 ± 0.9 g). Total body weights on each diet remained significantly lower (P < 0.0001) in UN offspring for the remainder of the study. Hypercaloric nutrition during postnatal life resulted in significantly (P < 0.0001) increased body weights, compared with control-fed animals, and by postnatal day 100, UN animals fed hypercalorically showed apparent catch-up growth to match the body weight of AD animals fed the control diet (Fig. 1Go). Body weight gain was increased in all IGF-I-treated animals (Fig. 2Go), and no difference in the response to body weight gain was observed between AD and UN offspring. However, daily weight gain was significantly lower in animals treated with IGF-I on hypercaloric nutrition as reflected by the significant (P < 0.05) diet and IGF-I interaction. At the end of the study, UN offspring were shorter than AD offspring in each treatment group and nose-anus lengths were significantly (P < 0.05) increased in all IGF-I-treated animals (Table 1Go). UN animals showed a significantly higher food intake on both diets, compared with AD animals. However, food intake was reduced (P < 0.005) by IGF-I treatment (Fig. 3Go). A significant statistical interaction was observed between programming and IGF-I treatment whereby reduction in food intake was more pronounced in UN animals following IGF-I treatment (P < 0.005).



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  		Figure 1. Postnatal growth curves of AD and UN offspring from weaning until commencement of IGF-I treatment [AD control diet ({circ}), AD hypercaloric diet ({blacktriangledown}), UN control diet ({square}), UN hypercaloric diet ({diamondsuit})]. Note the marked effect of hypercaloric nutrition on body weight of UN offspring and apparent catch-up growth to control-fed AD offspring by postnatal day 100 (n = 6 per group, data are mean ± SEM).

 


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  		Figure 2. Body weight gain (grams per day) during 14 d of IGF-I treatment. Programming effect NS, IGF-I treatment effect P < 0.0001, diet effect P < 0.05, diet and IGF-I treatment interaction P < 0.05 (n = 6 per group, data are mean ± SEM).

 

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  		Table 1. Body weight, length, and tissue weights of AD and UN offspring (age 190 ± 5 d) following 14 d treatment with IGF-I

 


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  		Figure 3. Food intake (kilocalories consumed per gram body weight per day) during 14 d of IGF-I treatment. Programming effect P < 0.0005, IGF-I treatment effect P < 0.0001, diet effect P < 0.0001, programming and IGF-I treatment interaction P < 0.005, programming and IGF-I treatment and diet interaction P < 0.05 (n = 6 per group, data are mean ± SEM).

 
Before onset of IGF-I therapy, SBP was markedly elevated (P < 0.0001) in UN offspring on the control diet, compared with AD offspring (AD control 121.84 ± 1.7 mm Hg, UN control 140.47 ± 2.12 mm Hg, AD hypercaloric 140.04 ± 2.63 mm Hg, UN hypercaloric 148.43 ± 1.59, P < 0.0001). The programming effect on hypertension was markedly amplified by postnatal exposure to hypercaloric nutrition (P < 0.0001). IGF-I treatment significantly reduced SBP in UN animals and in the group of AD offspring that had elevated blood pressure as a result of postnatal hypercaloric nutrition (Fig. 4Go).



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  		Figure 4. Change in SBP after 14 d of IGF-I treatment. Programming effect P < 0.0005, IGF-I effect P < 0.005, diet effect NS. There were no significant statistical interactions (n = 6 per group, data are mean ± SEM).

 
Blood plasma IGF-I concentrations were markedly increased in all IGF-treated offspring and the magnitude of the rise in plasma IGF-I was consistent in both AD and UN treatment groups (AD control saline 288 ± 23 ng/ml, AD control IGF-I 1318 ± 71 ng/ml, AD hypercaloric saline 330 ± 29 ng/ml, AD hypercaloric IGF-I 1308 ± 30 ng/ml, UN control saline 432 ± 31 ng/ml, UN control IGF-I 1253 ± 39 ng/ml, UN hypercaloric saline 391 ± 22 ng/ml, UN hypercaloric IGF-I 1291 ± 61 ng/ml, P < 0.0001). There was no significant effect of programming or diet on plasma IGF-I concentrations. Fasting plasma insulin levels were higher (P < 0.05) in UN offspring and were further elevated by hypercaloric nutrition (P < 0.0005). Treatment with IGF-I significantly lowered fasting plasma insulin concentrations (P < 0.005) in all offspring; this effect was most marked in the animals on hypercaloric nutrition (IGF-I treatment and diet interaction P < 0.005, Fig. 5AGo). Plasma glucose was not different between AD and UN offspring but was increased (P < 0.0001) by hypercaloric nutrition. IGF-I-treated animals showed markedly reduced plasma glucose concentrations (P < 0.0001) (Fig. 5BGo). Plasma leptin concentrations were higher (P < 0.005) in UN offspring and were increased (P < 0.0001) by hypercaloric diet. IGF-I treatment significantly lowered plasma leptin concentrations (P < 0.0005). As observed with insulin, there was a strong diet and IGF-I treatment interaction (P < 0.005, Fig. 6AGo) with plasma leptin levels being most markedly reduced by IGF-I treatment in offspring fed hypercalorically. Regression analysis revealed a strong positive relationship between plasma leptin and fasting insulin concentrations (r2 = 0.75, P < 0.0001). Retroperitoneal and gonadal fat pads were significantly larger in UN offspring (P < 0.05) and were further increased by hypercaloric nutrition in both AD and UN offspring (P < 0.0001). Treatment with IGF-I significantly reduced fat pad mass in all treated animals (P < 0.0001, Fig. 6Go, B and C). Regression analysis showed a highly significant positive relationship between fat mass and fasting plasma leptin (r2 = 0.765, P < 0.001, Fig. 6DGo).



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  		Figure 5. Fasting blood plasma insulin (A) and glucose concentrations (B) following 14 d of IGF-I treatment. Insulin: programming effect P < 0.05, IGF-I treatment effect P < 0.0001, diet effect P < 0.0005, diet and IGF-I treatment interaction P < 0.0005. Glucose: programming effect NS, IGF-I treatment effect P < 0.0001, diet effect P < 0.0001. There were no significant statistical interactions for fasting plasma glucose concentrations (n = 6 per group, data are mean ± SEM).

 


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  		Figure 6. A, Plasma leptin concentrations, B, retroperitoneal, C, gonadal fat pad weight (expressed as percent body weight) following 14 d saline (open bars) or IGF-I (closed bars) treatment, and D, the relationship between adipose mass and plasma leptin concentrations. Retroperitoneal fat: programming effect P < 0.05, IGF-I treatment effect P < 0.0001, diet effect P < 0.0001. Gonadal fat: programming effect P < 0.0001, IGF-I treatment effect P < 0.0001, diet effect P < 0.0001. Plasma leptin: programming effect P < 0.005, IGF-I treatment effect P < 0.0001, diet effect P < 0.0005, programming and diet interaction P < 0.05, diet and IGF-I interaction P < 0.005. There were no significant statistical interactions for retroperitoneal and gonadal fat pad weight (n = 6 per group, data are mean ± SEM).

 
Kidney weight was significantly (P < 0.0001) reduced in UN offspring (Table 1Go). AD and UN offspring fed hypercalorically had relatively lighter kidneys (P < 0.0001). Treatment with IGF-I significantly increased kidney weight (P < 0.0001). Heart weight was not different between AD and UN offspring but was reduced relative to body weight in animals fed hypercaloric nutrition. IGF-I treatment caused an increase in heart weight in all treated animals (P < 0.05). Liver weight was not different between AD and UN offspring and was not affected by diet. IGF-I-treated animals had lighter livers relative to body weight, compared with saline controls (P < 0.005). Spleen weight was not different between AD and UN offspring and was not altered by diet. However, treatment with IGF-I caused a significant increase in spleen weight in AD- and UN-treated animals (P < 0.0001). Relative brain weight in UN offspring was reduced, compared with AD offspring, and was lighter relative to body weight (P < 0.0001) in animals fed hypercalorically and/or treated with IGF-I. Adrenal weight was not different between UN and AD animals but was significantly (P < 0.0001) increased with IGF-I treatment (Table 1Go).

Plasma FFA concentrations were reduced in hypercalorically fed animals (P < 0.005, Table 2Go) but there was no effect of programming or IGF-I treatment. Plasma urea concentrations were markedly lower in UN offspring (P < 0.05, Table 2Go) and were decreased in all hypercalorically fed offspring (P < 0.0001). Treatment with IGF-I caused a significant reduction (P < 0.0001) in urea concentrations in all treated offspring. Plasma creatinine levels were not different between AD and UN offspring and were unaffected by diet. Treatment with IGF-I lowered (P < 0.0001) creatinine concentrations in all treated animals (Table 2Go).


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  		Table 2. Blood biochemistry analysis of AD and UN offspring (age 190 ± 5 d) following 14 d treatment with IFG-I

 
Alanine aminotransferase concentrations were significantly increased (P < 0.0001) in IGF-I-treated offspring but were not different between AD or UN offspring and were unaltered by hypercaloric nutrition (Table 2Go). Albumin concentrations were significantly (P < 0.05) lower in UN offspring, but there was no effect on diet or treatment. Calcium levels were higher (P < 0.05) in UN offspring, but there was no effect on diet or treatment. Plasma magnesium concentrations were markedly increased (P < 0.0001) with IGF-I treatment but were unaffected by diet and were not different between AD and UN offspring (Table 2Go).

Plasma IGFBPs were analyzed using nomenclature previously described (5, 31). The 38- to 44-kDa, 28- to 30-kDa, and 24-kDa bands represent IGFBP-3, IGFBP-1/2, and IGFBP-4, respectively. Analysis of plasma IGFBPs revealed that basal levels of all IGFBPs measured by ligand blot were elevated in UN offspring, compared with AD offspring. IGF-I treatment resulted in a 3- to 5-fold increase (P < 0.001) in IGFBP-3 in all IGF-I-treated animals (Fig. 7AGo). However, there was a diminished up-regulation of IGFBP-3 in UN animals indicated by a significant programming (P < 0.0001) and IGF-I treatment interaction (P < 0.0001). Postnatal hypercaloric nutrition significantly (P < 0.0001) reduced the IGFBP-3 band, compared with animals on the control diet, and reduced (P < 0.0001) the up-regulation of IGFBP-3 following IGF-I treatment; there was a significant (P < 0.05) programming, diet, and IGF-I treatment interaction. Interestingly although in UN animals the combined 38- to 44-kDa IGFBP-3 band showed impaired up-regulation following IGF-I treatment, analysis of the 38-kDa band alone showed an increase (P < 0.0001) of this band in UN animals, indicating a differential pattern of up-regulation of this form of IGFBP-3 in UN animals (Fig. 7BGo).



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  		Figure 7. Plasma IGFBPs as quantified by densitometry following ligand blotting analysis. A, IGFBP-3 (38–44 kDa): programming effect NS, IGF-I treatment effect P < 0.0001, diet effect P < 0.0001, programming and IGF-I treatment interaction P < 0.0001, diet and IGF-I treatment interaction P < 0.005, programming and IGF-I treatment and diet interaction P < 0.05. B, 38-kDa IGFBP-3: programming effect P < 0.0001, IGF-I treatment effect P < 0.0001, diet effect P < 0.0005. There were no significant statistical interactions for the 38-kDa IGFBP-3 band. C, IGFBP-1,-2 (28–30 kDa): programming effect NS, IGF-I treatment effect P < 0.0001, diet effect P < 0.05, programming and IGF-I treatment interaction P < 0.05. D, IGFBP-4 (24 kDa): programming effect P < 0.0001, IGF-I treatment effect P < 0.0001, diet effect P < 0.0005, programming and IGF-I treatment interaction P < 0.005, diet and IGF-I treatment interaction P < 0.05. Sample was 2 µl, n = 6 per group, data are mean ± SEM.)

 
Treatment with IGF-I significantly (P < 0.0001) increased (2- to 5-fold) the 28- to 30-kDa bands representing IGFBP-1/-2 and there was a diminished up-regulation of the IGFBP-1/-2 band following IGF-I treatment in UN animals, compared with AD animals (P < 0.05, Fig. 7CGo). Similarly, hypercaloric nutrition significantly reduced the increase in IGFBP-1/-2 following IGF-I treatment.

The 24-kDa band representing IGFBP-4 was significantly elevated in all UN animals (P < 0.0001, Fig. 7DGo) and was further amplified in all animals fed the hypercaloric diet (P < 0.0001). In an opposing pattern to the observation in other IGFBPs, a significant (P < 0.0001) down-regulation of IGFBP-4 was observed following IGF-I treatment. A significant (P < 0.001) programming and IGF-I treatment interaction revealed that IGFBP-4 was more markedly down-regulated in UN animals following IGF-I treatment, compared with AD animals. A significant diet and IGF-I treatment interaction was also observed with IGF-I treatment resulting in a lesser reduction in IGFBP-4 in hypercalorically fed animals, compared with those fed the control diet.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that fetal programming results in hyperinsulinemia, hyperleptinemia, hyperphagia, hypertension, and development of obesity in offspring during postnatal life (4, 32). Furthermore, the postnatal pathophysiology induced by fetal programming is markedly amplified by postnatal hypercaloric nutrition (4). We have also demonstrated in an earlier study that IGF-I treatment has lipolytic and antidiabetogenic effects (33) and IGF-I is well known to have vasodilatory functions in vivo and in vitro (20, 34). We therefore investigated in this study whether programming-induced metabolic and cardiovascular disorders in adult offspring can be alleviated by IGF-I therapy. Our results show that IGF-I treatment leads to a significant increase in body length, a marked reduction in food intake, decreased body fat mass, and normalization of blood pressure. Further endocrine responses include normalization of fasting insulin and glucose concentrations and a major reduction in plasma leptin concentrations. Our observation of a reduction in food intake despite the plasma leptin and insulin lowering effects of IGF-I invites a novel interpretation of IGF-I action. Firstly, IGF-I treatment may abolish the programming-induced leptin resistance at the leptin-hypothalamic circuitry and at the pancreatic adipoinsular feedback system. Secondly, IGF-I treatment may also ameliorate insulin resistance, both centrally and peripherally.

During treatment with IGF-I, we observed no significant difference in body weight response between AD and UN offspring, although a lower body weight gain was observed in all hypercalorically fed animals treated with IGF-I, compared with control-fed animals. As shown previously, treatment with IGF-I significantly reduced fasting plasma insulin and glucose concentrations in all treated animals (20, 35). A more pronounced decrease in plasma insulin concentrations was observed with IGF-I treatment in all animals fed a hypercaloric diet. However, in UN offspring animals (which were profoundly hyperinsulinemic) fed a hypercaloric diet, IGF-I treatment resulted in a return to basal fasting plasma insulin concentrations. In the present study, UN offspring were hyperphagic on both postnatal diets, compared with AD animals, confirming our previous observations. However, the significant decrease in plasma leptin concentrations following IGF-I treatment was associated with a decrease in food intake. More importantly however, the decrease in food intake following IGF-I treatment was more pronounced in offspring that were programmed to become obese and hyperphagic in adult life, which may explain the lower body weight gain observed in IGF-I-treated offspring fed hypercaloric nutrition. Although IGF-I treatment caused a significant reduction in food intake, weight gain was significantly increased in all IGF-I-treated animals. This may be a result of increased food conversion efficiency and significant increases in nitrogen balance and carcass nitrogen content following IGF-I treatment as reported previously (36). This would suggest that increased body weight as observed in the present study may be a result of altered partitioning of nutrients from fat to lean body tissue mass.

Data for a role of IGF-I in appetite regulation are limited, although early work by Tannenbaum et al. (37) showed that intracerebroventricular administration of IGFs resulted in a reduction in food intake. More recent work has also shown a reduction in food intake in tumor-bearing rats following infusion with either IGF-I or LR3-IGF-I (38). It is therefore tempting to speculate that our observation of reduced food intake following IGF-I treatment may be the result of the anorectic effect of IGF-I via its insulin-sensitizing effects and reduction of chronic hyperinsulinemia. Food intake was most markedly reduced in programmed animals fed hypercaloric nutrition; the same animals that showed the most marked decrease and normalization of fasting insulin and glucose concentrations following IGF-I treatment.

Our data on the lipolytic effect of IGF-I support results published previously (33, 39, 40, 41) and suggest that the effects of prolonged IGF-I treatment on adipose tissue are not insulin-like as reflected by increased lipolysis and decreased body fat mass. We propose that IGF-I treatment may reduce body fat mass via an inhibition of the lipogenic capacity of adipocytes and reduction of lipogenesis in adipose tissue via inhibition of insulin secretion. The lipolytic effects of IGF-I treatment were also concomitant with a significant decrease in fasting plasma leptin concentrations. This agrees with recent work in normal rats showing decreased plasma leptin and fat mass following constant infusion with rh-IGF-I for 6 d (18). It is unlikely that IGF-I acts via cross-reactivity with the insulin receptor. An insulin-like action would rather stimulate lipogenesis and thus increase fat pad weight and enhance leptin expression. It is also unlikely that IGF-I acts directly on adipose tissue via the IGF-I type 1 receptor. Rat adipose tissue lacks functional type 1 IGF receptors (41, 42) and IGF-I has been shown to have no effect on leptin secretion by mature adipocytes in vitro (43). Reduction of adipose tissue mass and suppression of leptin by IGF-I may be due to a reduction in circulating insulin leading to enhanced fat mobilization and nonesterified fatty acid oxidation as well as to increased gluconeogenesis from glycerol (18). However, in our study, although IGF-I infusion reduced adipose tissue weight, we observed no significant effect on plasma FFA concentrations. This may be due to increased triglyceride deposition and utilization in muscle tissue (author’s unpublished observations).

A further explanation for the metabolic effects of IGF-I observed in the present study may relate to the interactions between the leptin and insulin signaling networks (44), which may be disrupted as a result of fetal programming and further exacerbated by postnatal hypercaloric nutrition (32). Such dysregulation of the adipoinsular axis may contribute to the progression to insulin resistance and adipogenic diabetes (45, 46). Insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) co-ordinate essential effects of insulin/IGF on peripheral metabolism and ß cell function. Recent evidence suggests that impaired IRS-1 expression and downstream signaling events in adipocytes in response to insulin are associated with insulin resistance and the pentad of hypertension, hyperinsulinemia, dyslipidemia, obesity, and cardiovascular disease, known as syndrome X (7). IGF-I has been shown to inhibit insulin secretion from ß cells through an IGF-I receptor-mediated pathway (47, 48) and the IGF-I-IRS-2 signaling pathway has been proposed to be critical for postnatal ß cell function (49). It is tempting therefore to speculate that treatment with IGF-I may restore some of the functional feedback between the insulin signaling system and leptin action via modification of IRS-1 and IRS-2 and downstream signaling events.

Insulin resistance is often accompanied by hypertension, and obesity-induced hyperinsulinemia may induce alterations in sympathetic nervous system activity to increase blood pressure via vascular constriction. Because insulin-sensitizing agents have been shown to reduce blood pressure in obese, hypertensive subjects (50), it is possible that our observation of decreased SBP following IGF-I treatment may be a result of improved insulin sensitivity and glycemic control in conjunction with the known vasodilatory effects of IGF-I treatment (34). Further support for the antihypertensive effects of IGF-I as a result of improved insulin sensitivity stems from the observation that calcium and magnesium concentrations in circulation may regulate cellular responsiveness to insulin (51). In human hypertension, basal calcium levels are significantly elevated while basal magnesium concentrations are significantly decreased. Furthermore, elevated calcium or reduced magnesium concentrations are also observed in clinical states linked to hypertension, such as obesity and type 2 diabetes (52). Our observations of elevated calcium in hypertensive UN animals and a significant increase in plasma magnesium after IGF-I treatment agrees with these findings and suggests that IGF-I treatment may alleviate insulin resistance and reduce hypertension in part by changing the calcium/magnesium ratio in plasma.

The highly significant increase in kidney weight with IGF-I treatment may also be an important factor in reduction of SBP via changes in renal plasma flow and glomerular filtration rate. Given recent in vitro observations (22, 53), it is tempting to speculate that IGF-I treatment may also reduce blood pressure by down-regulating the local RAS and limiting angiotensin II formation through mediation of the angiotensin type 1 receptor (23). Heart weight in all IGF-I-treated animals was significantly increased, which may reflect myocyte growth and improved contractility. Others have shown cardiac hypertrophy and increased left ventricular mass following IGF-I treatment (20, 54, 55). Importantly, IGF-I treatment reduced SBP only in animals that were hypertensive as a result of fetal programming or postnatal hypercaloric nutrition, and systolic blood pressure in normotensive animals remained unaltered.

Some effects of IGF-I treatment on improving insulin sensitivity and ameliorating the postnatal pathophysiology following fetal programming may be mediated by changes in circulating IGFBPs. Previous work by our group (5) has shown that circulating IGFBPs are differentially regulated as a result of fetal programming. However, data on the effect of IGF-I therapy on IGFBPs in postnatal life following fetal programming are limited. In the present study, fetal programming led to an increase in circulating levels of IGFBP-1/-2 and -4 concentrations as shown by Western ligand blotting. Similar results have been observed before in the serum of growth-retarded fetuses, compared with control fetuses (25, 26). The IGFBP-1/-2 doublet has also been previously shown to be increased in growth-retarded fetuses using a model of uterine artery ligation in the rat; because serum immunoreactive IGFBP-2 was unchanged among the groups, it was suggested that IGFBP-1 accounted for the increase in doublet intensity (56). Elevated IGFBP-1 is normally associated with poor glycemic control and implicated in the pathogenesis of type 2 diabetes because a rise in IGFBP-1 has been related to inadequate portal delivery of insulin (57, 58, 59). Previous work by our group (60) has shown that plasma levels of IGFBP-3 are increased 2-fold following treatment with h-IGF-I in the GH-deficient dwarf rat. Work by others (61) has shown a highly significant increase in IGFBP-3 following IGF-I therapy in the normal rat. The mechanism underlying the preferential up-regulation of the 38-kDa IGFBP-3 band in UN animals following IGF-I treatment is still to be elucidated but may relate to changes in phosphorylation or glycosylation of IGFBP-3 (62).

Fetal programming resulted in a significant elevation in circulating IGFBP-4 levels, which were amplified by postnatal hypercaloric nutrition. Treatment with IGF-I decreased circulating IGFBP-4 in all treated animals; moreover, IGF-I treatment was more effective in reducing IGFBP-4 concentrations in those animals that had become obese as a result of fetal programming and hypercaloric nutrition. This observation is not surprising because IGFBP-4 appears to inhibit IGF-I action under most, if not all, experimental conditions (63). It is tempting to speculate that IGF-I treatment causes activation of IGFBP-4 proteases and may result in the degradation and inactivation of IGFBP-4 as reported by others (64, 65, 66). The increase in circulating IGFBP-1, -2, and -3 and the decrease in IGFBP-4 with IGF-I treatment may represent a mechanism of increasing IGF-I activity at the different target tissues discussed above.

In conclusion, our animal model of fetal programming by maternal undernutrition during pregnancy results in profound hyperphagia, obesity, hypertension, hyperinsulinemia, and hyperleptinemia during adult life. Postnatal hypercaloric nutrition amplifies the metabolic and cardiovascular pathophysiology consistent with the clinical setting of syndrome X (7). Treatment with IGF-1 at an adult age showed a significant increase in body length, a marked reduction in food intake and body fat mass, and normalization of blood pressure. Further intriguing findings include reduction of fasting plasma insulin and leptin concentrations. Thus, IGF-1 treatment may alleviate insulin and leptin resistance and improve obesity, hyperphagia, and hypertension by differential effects on IGF-I receptor-signaling pathways or downstream signaling networks including the IRS and RAS. IGF-I treatment may also restore functional interactions between insulin and leptin following perturbations of the hypothalamic circuitry that controls food intake and of the adipoinsular axis as a result of fetal programming.


   Acknowledgments
 
We thank Christine Keven, Andrzej Surus, and Janine Street for their expert technical assistance.


   Footnotes
 
This work was supported by the Health Research Council of New Zealand and the National Child Health Research Foundation.

Abbreviations: AD, Ad libitum; IGFBP, IGF-binding protein; IRS, insulin receptor substrate; RAS, renin-angiotensin system; rh-IGF-1, recombinant human IGF-1; SBP, systolic blood pressure; UN offspring, offspring from mothers that were undernourished throughout pregnancy.

Received December 15, 2000.

Accepted for publication May 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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