Superimposition of Postnatal Calorie Restriction Protects the Aging Male Intrauterine Growth- Restricted Offspring from Metabolic Maladaptations

Abstract

Intrauterine growth restriction (IUGR) results in dysregulated glucose homeostasis and adiposity in the adult. We hypothesized that with aging, these perturbations will wane, and superimposition of postnatal growth restriction (PNGR) on IUGR [intrauterine and postnatal growth restriction (IPGR)] will reverse the residual IUGR phenotype. We therefore undertook hyperinsulinemic-euglycemic clamp, energy balance, and physical activity studies during fed, fasted, and refed states, in light and dark cycles, on postweaned chow diet-fed more than 17-month aging male IUGR, PNGR, and IPGR vs. control (CON) rat offspring. Hyperinsulinemic-euglycemic clamp revealed similar whole-body insulin sensitivity and physical activity in the nonobese IUGR vs. CON, despite reduced heat production and energy expenditure. Compared with CON and IUGR, IPGR mimicking PNGR was lean and growth restricted with increased physical activity, O₂ consumption (VO₂), energy intake, and expenditure. Although insulin sensitivity was no different in IPGR and PNGR, skeletal muscle insulin-induced glucose uptake was enhanced. This presentation proved protective against the chronologically earlier (5.5 months) development of obesity and dysregulated energy homeostasis after 19 wk on a postweaned high-fat diet. This protective role of PNGR on the metabolic IUGR phenotype needs future fine tuning aimed at minimizing unintended consequences.

Developmental origins of health and adult disease hypothesis (DoHAD) states that adverse prenatal and early postnatal nutritional environments increase the risk for adult onset obesity, diabetes, and associated metabolic syndrome (1).

Consistent with DoHAD, animal models of prenatal nutritional restriction, especially global calorie restriction (2–6), low protein maternal diet (7, 8), and uterine artery ligation (9–13), have detected glucose and lipid metabolic abnormalities in low birth weight offspring that experience subsequent accelerated catch-up growth.

A recent “predictive adaptive hypothesis” along with the “match-mismatch theory” extended DoHAD by stating that the structure and function of fetal organs are programmed in response to prenatal malnutrition to confer an advantage to handle the future postnatal environment (14, 15). When the postnatal environment fails to match the experienced prenatal environment, maladaptation occurs, resulting in adult onset chronic diseases. In contrast, if the postnatal environment matches the fetal prediction, the offspring is protected from metabolic syndrome (14, 15).

The “lean” phenotype with glucose tolerance has been reported in mice on chow (16, 17) or hypercaloric diets (18), when postnatal calorie or protein restriction (19) reversed the development of insulin resistance. Male mice on chow or cafeteria diets that were exposed to postnatal protein restriction demonstrated prolonged longevity compared with mice with fetal growth restriction and postnatal catch-up growth (20, 21). However, in these studies, the mechanisms responsible for the lean phenotype were not investigated (20, 21). Furthermore, the studies were pursued in younger adult offspring ranging in age from 5 to 12 months (16–18). Previous investigations in rats employing early protein restriction demonstrated that insulin resistance is encountered during aging at 21 months of life (7), whereas others have contended that aging in the intrauterine growth restriction (IUGR) offspring continues to demonstrate an insulin-sensitive state (22).

Hence, we undertook the present study to test the hypotheses that 1) aging partially ameliorates the overt metabolic perturbations and obesity of the IUGR adult offspring, and 2) a match between pre- and postnatal nutrition will further protect the aging IUGR male offspring from developing metabolic abnormalities and obesity.

Materials and Methods

Sprague Dawley rats (Charles River Laboratories, Hollister, CA) were housed in individual cages, exposed to 12-h light, 12-h dark cycles at 21–23 C, and allowed ad libitum access to standard rat chow (Laboratory Rodent Diet 5001: protein 23%, fat 4.5%, fiber 6%, ash 8.0%, and minerals 2.5%; LabDiet, Rancho Cucamonga, CA). The National Institutes of Health guidelines for care and maintenance of animals were followed as approved by the Animal Research Committee of the University of California, Los Angeles.

Maternal calorie restriction model

Pregnant Sprague Dawley rats received 50% of their daily food intake beginning from d 11 through d 21 of gestation, causing caloric restriction during mid to late gestation, compared with their control (CON) counterparts, who received ad libitum rat chow. Both groups had ad libitum access to drinking water. At birth, the litter size was culled to six. Four groups were created by cross-fostering all pups on d 2 of life, with control mothers rearing control pups or prenatally calorie-restricted pups (IUGR) and pre- and postnatally calorie-restricted mothers rearing prenatally calorie-restricted pups [intrauterine and postnatal growth restriction (IPGR) or control postnatal growth restriction (PNGR)] pups (Fig. 1) (23, 24). At d 21, the male pups from all four groups were weaned from the respective mothers and maintained on the same rat chow ad libitum. At 17 months of age, their phenotype was examined.

Fig. 1.

Study design. Scheme of the study design demonstrates four experimental groups obtained by cross-fostering postnatal rat pups in all four groups on d 2 of life. 1) CON rats, i.e. CON mothers rearing CON pups; 2) IUGR pups, i.e. CON mothers rearing prenatally calorie-restricted pups; 3) IPGR rats, i.e. 50% prenatally and postnatally calorie-restricted mothers rearing prenatally caloric-restricted pups; and 4) PNGR rats, i.e. 50% calorically restricted mothers rearing CON pups.

Energy intake, body weight, and white adipose tissue (WAT)

Food intake and body weight of male rats at 17 months of age were measured weekly over a 24-h period, and four readings were averaged. Energy intake was assessed based on the conversion factor of 4.07 kcal/g 5001 rat chow food intake (values calculated are based on ingredient analysis by manufacturer; Harlan Laboratories, Madison, WI). Visceral WAT was mechanically isolated and weighed. Energy intake and WAT were expressed as a percent of body weight.

Plasma assays

Plasma glucose concentration was measured by a HemoCue glucose 201 analyzer. Plasma insulin was measured using a Rat Insulin ELISA kit (Linco Research, Billerica, MA).

Hyperinsulinemic euglycemic clamp

One week before the study, under isoflurane (2–3.5% vapor concentration in O₂), two internal jugular vein catheters extending to the right atrium, and a right carotid artery catheter extending to the level of the aortic arch, were implanted. Analgesics (ketoprofen) and antibiotics (ampicillin) were given daily for the first 48 h after surgery. All rats were fasted for 16 h before the study. The steady state in rats was achieved by a bolus injection of (3-³H) glucose (5-μCi bolus) followed by the infusion of (3-³H) glucose (0.05 μCi/min) for 90 min. This time point is referred to as 0. At time 0, hyperinsulinemic-euglycemic clamp study was started, and rats received an infusion of insulin (Novolin R, regular human insulin; Novo Nordisk, Bagsværd, Denmark) at 4 mU/kg·min and (3-³H) glucose at 0.2 μCi/min simultaneously. A variable infusion of 50% glucose solution was started and adjusted to clamp the plasma glucose concentration between 100 and 120 mg/dl. To assess the insulin-stimulated glucose uptake [2-deoxyglucose (2-DG)] in individual tissues, 10 μCi of 2-deoxy-D-[1-¹⁴C] glucose (PerkinElmer, San Jose, CA) were infused as a bolus 45 min before the end of the clamp study. At the end of the clamp study, rats were euthanized with pentobarbital sodium (50 mg/ml; 100 mg/kg) injection.

Analytical procedure

Plasma (3-³H) glucose-specific radioactivity was measured using Somogyi method as described previously (12). The glucose disposal rate (R_d) at steady state was determined by dividing the infusion rate of labeled glucose by the specific activity at the same time point. Endogenous glucose production was determined by subtracting the unlabeled glucose infusion rate (GIR) from R_d.

Accumulation of phosphorylated derivatives of radioactive 2-DG by tissue

Total and free 2-DG radioactivity in tissue samples that included liver, WAT, soleus, extensor digitorum longus, and gastrocnemius muscles were assessed, and 2-DG-phosphate was calculated as described previously (25).

Indirect calorimetry

Energy expenditure in rats was measured using the Oxymax Lab Animal Monitoring System (Columbus Instruments, Columbus, OH). Seventeen-month-old male rats were individually housed and allowed to acclimate for at least 24 h in experimental cages with free access to food and water (26–28). O₂ consumption (VO₂), heat production, respiratory exchange ratio (RER), and physical activity were recorded during light and dark cycles over a 24-h period during the fed state. Energy expenditure in rats under the fasting condition constituted monitoring during the last 2 h of a 16-h overnight fast. The refed condition was monitored over a 24-h period after ad libitum access to food after the overnight fast. Energy expenditure in rats under resting conditions (resting energy expenditure) was based on a 24-h period measurement by indirect calorimetry and was computed in the light cycle. An average of at least five measurements (VO₂, RER, and heat production) was obtained when rats were not consuming food or water and with none to minimal activity (zero to three counts/10-min epoch) (29–31).

Physical activity

Spontaneous physical activity was classified as total activity, horizontal movement as ambulation that breaks the laser beam in the x-axis, and vertical movement as rearing that breaks the beam in the z-axis. Data were analyzed using the OxymaxWin version 4.0 software package.

High-fat diet (HFD)

A separate set of CON and IPGR rats was fed with either a HFD (Harlan Teklad TD 06414: protein 23.5%, fat 34.3%, and carbohydrate 27.3%) or regular chow diet beginning with weaning at 21 d of age. The HFD or CON feeding lasted for 19 wk. Energy intake, body weight, and WAT weights were measured. Energy intake was assessed based on the conversion factor of 5.1 kcal/g T.06414 rat (60%/fat) chow food intake (values calculated are based on ingredient analysis by manufacturer; Harlan Laboratories). WAT weights were expressed as a percent of body weight. VO₂, heat production, and physical activity were assessed over a 24-h period by indirect calorimetry.

Western blot analysis

Western blottings using skeletal muscle homogenates, including soleus, extensor digitorum longus, and gastrocnemius, were performed as previously described (24). Primary antibodies anti- Sirtuin1 (Sirt1) (Millipore, Billerica, MA), anti-AMPKα and p-adenosine monophosphate kinase C (AMPK)α (Cell Signaling Technology, Danvers, MA), and anti-protein kinase (PKC)ζ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used to detect specific proteins in skeletal muscle. antivinculin antibody (Sigma Chemical Co., St. Louis, MO) was used as internal CON.

Data analysis

Data are expressed as mean ± se. For hyperinsulinemic-euglycemic clamp and energy expenditure studies (including the HFD study), two-way repeated measures ANOVA analysis, with group treated as between subject factor and treatment (basal vs. clamp, light and dark for energy expenditure) as within subjects factor followed by Tukey's post hoc analysis. For energy and water intake, three factors of group, light (dark) cycle, and metabolic settings (refed and fed) were compared using three-way ANOVA analysis (split-plot factorial), with light (dark) cycle and metabolic settings (fed or refed) being two factors as within subjects factor and group as between the subjects factor. Tukey-Krammer pairwise comparison was employed for post hoc analysis. For the HFD study, Student's t test was employed to compare body weight and WAT between CON-HFD and IPGR-HFD groups.

Results

Postweaning chow diet studies

Body weights, WAT weights, and plasma glucose and insulin concentrations

Birth weight of male pups born to calorie-restricted mothers (IUGR, 6.5 ± 0.1 g) was 82% of pups born to CON mothers (CON, 7.9 ± 0.1 g; P < 0.05). By d 50 of age, IUGR “caught up” to CON (92%; P > 0.05), whereas IPGR and PNGR remained lighter than CON, at approximately 70% of age matched CON (P < 0.05). By 17 months, PNGR caught up to CON, but IPGR remained growth restricted at 77% of CON (P < 0.05). The body weights of IUGR (P > 0.05) and PNGR (P > 0.05) were similar to that of CON. Reflecting the body weight, WAT depicted as a percent of body weight in IUGR was no different from CON. In contrast, IPGR exhibited a significant reduction in percent of WAT/body weight vs. both CON and IUGR (see figure 3 below). Plasma glucose and insulin were not different between the four groups (Table 1).

Table 1.

Body weights and serum metabolites

	BW 50 d (g)	BW 17 months (g)	Glucose (mmol/liter) (17 months)	Insulin (ng/ml) (17 months)
CON	276.2 ± 5.1	904.0 ± 51.2	8.8 ± 0.4	2.1 ± 0.4
IUGR	253.5 ± 5.1a	8840.0 ± 45.1	9.0 ± 0.8	2.2 ± 0.4
IPGR	198.3 ± 1.5b	695.5 ± 22.5a	8.7 ± 0.5	2.1 ± 0.3
PNGR	183.7 ± 4.5c	816.3 ± 56.2	8.8 ± 0.7	2.4 ± 0.2

Body weight (BW) of four experimental groups at 50 d and 17 months. At 50 d, IUGR was lighter than CON (^a), IPGR was lighter than CON and IUGR (^b), and PNGR was even lighter than CON, IUGR, and IPGR (^c), n = 6 in each group. By 17 months, IPGR was significantly smaller than CON (P < 0.05), whereas the BW of IUGR (P > 0.05) and PNGR (P > 0.05) were similar to that of CON. Serum metabolites of four experimental groups at 17 months are shown. Serum glucose and insulin were not significantly different between the four groups (P > 0.05): CON (n = 8), IUGR (n = 7), IPGR (n = 6), and PNGR (n = 8).

^aP < 0.05 vs. CON.

^bP < 0.05 vs. CON and IUGR.

^cP < 0.05 vs. CON, IUGR and IPGR.

Glucose kinetics

IUGR was insulin sensitive and both PNGR and IPGR had no effect on glucose R_d, GIR and hepatic glucose production (HGP) at 17 months of age. Despite IPGR's lighter body weight, no significant differences existed in R_d among the four groups in the basal state. Insulin-stimulated R_d, HGP, and GIR were also similar among the four groups. However, significant differences were detected in R_d between basal and hyperinsulinemic-euglycemic clamped states within the same group, that is, insulin-stimulated R_d was significantly higher compared with basal R_d in all four groups (P < 0.05 each) (Fig. 2A). Basal and insulin-stimulated HGP was similar among the four groups. In response to an exogenous insulin infusion, CON, IUGR, IPGR, and PNGR demonstrated 30% reduction in HGP (Fig. 2B). Steady state GIR required to maintain euglycemia during hyperinsulinemic-euglycemic clamp were similar in four groups (Fig. 2C). These results suggest that whole-body insulin sensitivity of IUGR, IPGR, and PNGR was similar to CON.

Fig. 2.

Hyperinsulinemic-euglycemic (HI-EG) clamp. HI-EG clamp results are shown in the four experimental groups: CON (n = 8), IUGR (n = 7), IPGR (n = 6), and PNGR (n = 8). A, During HI-EG clamp, glucose use (R_d) (shown in black bars) increased significantly compared with basal R_d (shown in light bars) in CON, IUGR, IPGR, and PNGR [two-way repeated measures ANOVA, F_(3,1) = 1.54, a, P < 0.001; Tukey's post hoc analysis: a, P < 0.05]. Basal R_d and R_d during HI-EG clamp were not significantly different between the four experimental groups (P > 0.05). Statistical methods: two-way repeated measures ANOVA analysis, compares the groups as between subject factor and treatment (basal vs. clamp) as within subject factor. B, HGP during HI-EG clamp (shown in black bars) decreased significantly compared with basal HGP (shown in light bars) in CON, IUGR, IPGR, and PNGR two-way repeated measures ANOVA [F_(3,1) = 31.993, P < 0.001; Tukey's post hoc analysis: a, P < 0.05]. Basal HGP and HGP during HI-EG clamp were not significantly different between the four experimental groups (P > 0.05). C, GIR required to maintain euglycemia during a HI-EG clamp did not show significant changes in CON, IUGR, IPGR, and PNGR (P > 0.05, one-way ANOVA analysis). D, 2-DG uptake in skeletal muscle (SOL, Soleus; EDL, extensor digitorum longus; GAS, gastrocnemius), liver (LIV), and WAT is shown during a HI-EG clamp for all groups. SOL: 2-DG uptake increased in IPGR (IPGR vs. CON and PNGR; *‡, P < 0.05). EDL: 2-DG uptake increased in IPGR (IPGR vs. CON, IUGR, and PNGR; *‡#, P < 0.05). GAS: 2-DG uptake increased in IPGR (IPGR vs. CON and IUGR; *#, P < 0.05) and in PNGR (PNGR vs. CON; *, P < 0.05). There was no change in 2-DG uptake in LIV and WAT between CON, IUGR, IPGR, and PNGR (P > 0.05).

Tissue glucose uptake

IUGR remained insulin sensitive, whereas IPGR revealed increased skeletal muscle 2-DG uptake. As shown in Fig. 2D, insulin-stimulated glucose uptake by skeletal muscle was significantly increased in IPGR compared with CON, IUGR, and PNGR. Specifically the 2-DG uptake in the soleus (type 1 fibers; slow twitch muscle) was approximately 2-fold over CON (P < 0.05) and PNGR (P < 0.05). Similarly, in IPGR, insulin-stimulated 2-DG uptake was increased in extensor digitorum longus (mainly fast twitch fibers) and gastrocnemius (mixture of slow and fast twitch muscle fibers) by approximately 2-fold over CON (P < 0.05 each) and IUGR (P < 0.05 each). Although in IPGR insulin-stimulated 2-DG uptake by extensor digitorum longus was 2-fold over PNGR (P < 0.05), in PNGR, insulin-stimulated gastocnemius 2-DG uptake was 1.3-fold over CON (P < 0.05). No difference between IUGR and CON was observed in the case of soleus, extensor digitorum longus, and gastrocnemius. There were no intergroup differences noted in either liver or WAT (Fig. 2D).

Skeletal muscle protein expression studies

Skeletal muscle (soleus, extensor digitorum longus, and gastrocnemius combined) Sirt1, total AMPK, pAMPK, and total PKCζ protein concentrations were no different in the four experimental groups (Table 2), although pAMPK in IUGR trended toward an increase vs. that of CON.

Table 2.

Skeletal muscle protein expression profile

	CON	IUGR	IPGR	PNGR
Sirt1	100.0±7.8	95.0±8.7	88.8±7.3	90.2±9.6
PKCζ	100.0±1.8	92.1±2.3	98.2±2.6	104.3±1.8
AMPK	100.0±5.2	98.3±9.4	110.8±9.7	118.9±11.2
PAMPK	100.0±19.5	122.7±12.5	110.8±19.0	111.7±25.5

Energy balance

IUGR demonstrated reduced whole-body energy expenditure, whereas PNGR and IPGR enhanced whole-body energy expenditure in aging rats.

Energy intake

Energy intake during light and dark cycles was similar among the four groups during fed and refed states (data not shown). When normalized to body weight, regardless of the fed or refed state, IPGR consumed more energy vs. CON and IUGR (P < 0.05 each, three-way ANOVA), whereas PNGR consumed more energy than IUGR (P < 0.05, three-way ANOVA) (Fig. 3A). Significantly more energy was consumed during dark cycle compared with light cycle in each group in both fed and refed states. Water intake was similar among four groups with or without normalization to body weight (data not shown).

Fig. 3.

Energy intake, %WAT weight/body weight (BW), and BMR. Energy intake (EI) measured over 24 h during indirect calorimetry and apportioned to light and dark cycles are shown in fed and refed states for CON (n = 8), IUGR (n = 7), IPGR (n = 6), and PNGR (n = 8). Experimental groups, light-dark cycle, and fed/refed states were compared using three-way ANOVA analysis (split-plot factorial) followed by Tukey's post hoc analysis. Ratios of WAT to BW (%) in CON, IUGR, IPGR, and PNGR were calculated and compared, n = 6 in each group. A, Energy intake (EI) normalized to BW increased in IPGR (*#, P < 0.05, IPGR vs. CON and IUGR, respectively) and PNGR (#, P < 0.05, PNGR vs. IUGR). Differences between light and dark cycles were assessed using tests of simple effects, which found a significant increase in the dark cycle in IPGR and PNGR compared with their respective light cycle (Δ, P < 0.05). B, Percent of WAT/BW. IPGR demonstrated a decreased WAT weight compared with CON and IUGR (*#, P < 0.05, IPGR vs. CON and IUGR, respectively), there were no changes in WAT weight between CON, IUGR, and PNGR (P > 0.05). C and D, Resting energy expenditure and BMR. Energy metabolic parameters obtained during 24 h of indirect calorimetry in CON (n = 8), IUGR (n = 7), IPGR (n = 6), and PNGR (n = 8). Resting energy expenditure was an average of at least five measurements [VO₂, RER (data not shown), and heat production (HP)] in the absence of food or water consumption during none to minimal activity (zero to three counts/10-min epoch) in the light cycle obtained during 24 h of indirect calorimetry. All comparisons were made by one-way ANOVA followed by Tukey's post hoc analysis. C, VO₂. IUGR exhibited significantly reduced VO₂ compared with that of CON, IPGR, and PNGR (*†‡, P < 0.05 vs. CON, IPGR, and PNGR). D, Heat Production (HP). IUGR demonstrated significantly reduced HP (*, P < 0.05 vs. CON).

Energy expenditure (VO₂)

IUGR demonstrates decreased basal metabolic rate (BMR) in the absence of obesity, whereas PNGR and IPGR remain unchanged. During the resting state, in the IUGR, VO₂ (∼50–60% of CON, IPGR, and PNGR; P < 0.05) (Fig. 3C) and heat production decreased (∼60% of CON, P < 0.05; IPGR and PNGR, P > 0.05 each) (Fig. 3D), whereas intergroup RER was unchanged (P > 0.05, data not shown). In contrast, during the resting state, VO₂ (Fig. 3C), RER (data not shown), and heat production (Fig. 3D) in IPGR and PNGR were similar to CON.

IUGR exhibited decreased energy expenditure (VO₂) in fed, fasting, and refed states. VO₂ was reduced during the fed state in light cycle in IUGR vs. the other three groups (∼60–70% of CON, IPGR, and PNGR; P < 0.05) (Fig. 4Ai). When compared with IPGR and PNGR, a decrease in VO₂ also occurred in IUGR in light cycle during fasting (IUGR vs. CON, P > 0.05; but 67% of IPGR and PNGR, P < 0.05 each) and refed states (IUGR is 66% of IPGR, 73% of PNGR; P < 0.05 each) (Fig. 4A, ii and iii). During dark cycle, VO₂ of IUGR decreased in fed (IUGR is 68% of IPGR and PNGR; P < 0.05 each) and refed states (IUGR is 63% of IPGR, 74% of PNGR; P < 0.05 each) vs. IPGR and PNGR (Fig. 4A, i and iii).

Fig. 4.

Energy expenditure and physical activity. A–C, Energy metabolic parameters obtained during 24 h of indirect calorimetry in light and dark cycles are shown in fed (i), fasting (ii), and refed (iii) states for the four experimental groups. All comparisons between groups were made by two-way repeated measures ANOVA followed by Tukey's post hoc analysis. A, VO₂ in IUGR decreased during light cycle in fed (IUGR vs. CON, IPGR, and PNGR; *†‡, P < 0.05) (i), fasting (IUGR vs. IPGR and PNGR; †‡, P < 0.05) (ii), and refed states (IUGR vs. IPGR and PNGR; †‡, P < 0.05) (iii), being no different in the IUGR between the three states of fasting, fed vs. refed states. VO₂ in IPGR increased during the dark cycle in the fed state (i) and in dark and light cycles in the refed state (iii) (IPGR vs. CON and IUGR; §ϵ, P < 0.05) with no change with fasting (ii). VO₂ in PNGR increased during the dark cycle in the fed state (PNGR vs. CON and IUGR; §ϵ, P < 0.05) (i) and in the refed state (PNGR vs. CON; §, P < 0.5) (iii) with no change during the fasting state (ii). B, Heat production (HP) (kcal/h) in IUGR decreased during the light cycle in fed state (IUGR vs. CON; *, P < 0.05) (i) and during dark cycle in the refed state (IUGR vs. CON and IPGR; §μ, P < 0.05) (iii). No difference was noted in IPGR and PNGR compared with the respective CON. C, Physical activity (PA) measured during the 24-h period by indirect calorimetry was apportioned to the light and dark cycles and shown in fed (i), fasting (ii), and refed states (iii) for the four experimental groups, namely CON (n = 8), IUGR (n = 7), IPGR (n = 6), and PNGR (n = 8). All comparisons between groups employed the two-way repeated measures ANOVA followed by Tukey's post hoc analysis. Total activity in IUGR did not change significantly in fed (i), fasting (ii), and refed (iii) states during both the light and dark cycles, compared with CON (P > 0.05). Total activity increased in IPGR in the fed state during the dark cycle (IPGR vs. CON; §, P < 0.05) (i), in fasted state during the light cycle (IPGR vs. CON, IUGR, and PNGR; *#‡, P < 0.05) (ii), and in the refed state (IPGR vs. CON and IUGR; ϵ§, P < 0.05) (iii). Total activity in PNGR did not change significantly in fed (i), fasting (ii), and refed (iii) states during both the light and dark cycles compared with CON (P > 0.05).

IPGR expressed increased energy expenditure (VO₂) in fed, fasting, and refed states. VO₂ in fed state during dark cycle of IPGR increased vs. that of CON and IUGR (P < 0.05 each). During fasting state, VO₂ of IPGR increased vs. that of IUGR (P < 0.05). In refed state during light cycle, IPGR demonstrated an increase in VO₂ vs. that of CON (P < 0.05). During dark cycle as well, VO₂ of IPGR increased over that of CON and IUGR (P < 0.05 each) (Fig. 4A, i–iii).

PNGR demonstrated an increase in energy expenditure (VO₂) in fed, fasting, and refed states. VO₂ of PNGR in fed state during dark cycle increased over that of CON (P < 0.05) and IUGR (P < 0.05). During refed state as well in dark cycle, VO₂ of PNGR increased over that of IUGR (P < 0.05) (Fig. 4A, i–iii).

Respiratory exchange ratio

No intergroup differences were detected in RER during fed, fasting, or refed states. However, in all four experimental groups, fasting decreased RER from 0.9 of fed and refed states to 0.7. This change supported a switch in the use of fuel from carbohydrates in fed and refed states to fat in the fasting state (Supplemental Fig. 1A, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org).

Heat production

IUGR reduced heat production. Heat production by IUGR during fed state in light cycle was reduced to that of CON (P < 0.05). During the refed state in dark cycle, heat production by IUGR was also diminished vs. that of CON and IPGR (P < 0.05 each) (Fig. 4B, i–iii).

Physical activity

IUGR was not inactive, whereas IPGR and PNGR expressed enhanced physical activity.

Total activity

Total activity of IPGR during fed state in dark cycle increased over that of CON (P < 0.05). During fasting state, total activity of IPGR increased 2.4-fold of CON and only 1.6-fold of IUGR and PNGR (P < 0.05 each). During refed state in dark cycle as well, IPGR demonstrated increased total activity vs. CON and IUGR (P < 0.05 each) (Fig. 4C, i–iii).

Ambulation (horizontal)

During fed and refed states in dark cycle, IPGR demonstrated increased ambulation compared with CON (P < 0.05) (Supplemental Fig. 1B, i–iii). Similar to IPGR, during fed state in dark cycle, PNGR exhibited more ambulation compared with CON and IUGR (P < 0.05 each), respectively. During fasting, IPGR demonstrated a 2.6-fold increase in ambulation vs. CON, IUGR, and PNGR (P < 0.05 each) (Supplemental Fig. 1B).

Rearing (vertical)

The rearing of IPGR during fed state in dark cycle increased over that of CON (P < 0.05). Similar increases in rearing of IPGR during refed state in light cycle were also observed, reflected as 2.5-fold of IUGR (P < 0.05). During the refed state in dark cycle, rearing of IPGR increased over that of CON, IUGR, and PNGR (P < 0.05 each) (Supplemental Fig. 1C).

Postweaning HFD studies

Postweaning HFD intake in IPGR and CON

IPGR raised on HFD was relatively protected from obesity seen as lower body weight and lower WAT expressed as a percent of body weight vs. the CON counterpart raised on a HFD (P < 0.05) (Fig. 5, A and B). IPGR and CON raised on HFD were no different in energy (Fig. 5C) and water intake (data not shown), although VO₂ in light and dark cycles (P < 0.05) (Fig. 5D), heat production (dark cycle, P < 0.05) (Fig. 5E), and physical activity (total, P < 0.05) in dark cycle were increased in IPGR vs. CON (Fig. 5F). Although no change in RER was evident, an increase in ambulation and rearing was also observed in dark cycle in IPGR vs. CON (Supplemental Fig. 2).

Fig. 5.

Effects of HFD on CON and IPGR groups. Rats were fed a HFD containing 60% of calories derived from fat for 19 wk from weaning (postnatal d21) until 160 d (5.5 months of age), n = 6 in each group. A, Body weights (BW). IPGR was protected against the development of HFD-induced postweaned obesity by demonstrating a significant reduction in BW compared with CON; *, P < 0.05 compared with CON, Student's t test. B, Percent of visceral WAT/BW decreased in IPGR-HFD compared with CON-HFD; *, P < 0.05 compared with CON, Student's t test. C, Energy intake (EI)/BW did not change significantly between CON-HFD and IPGR-HFD during both the light and dark cycles (P > 0.05, two-way repeated measures ANOVA). D and E, Twenty-four hours of indirect calorimetry, similar to that seen in CON and IPGR (Fig. 4, A–C) in light and dark cycles, is shown in the fed state (n = 6 in each group). All comparisons between the two assessments of energy metabolism apportioned to the groups were performed by two-way repeated measures ANOVA followed by Tukey's post hoc analysis. D, VO₂ in IPGR-HFD increased in both light and dark cycles vs. the respective CON-HFD (*, P < 0.05). E, Heat production (HP) in IPGR-HFD increased during the dark cycle vs. that of CON-HFD (*, P < 0.05). F, Total activity in IPGR-HFD was significantly increased compared with CON-HFD during the dark cycle (*, P < 0.05).

Summary of our results is schematically depicted in Table 3.

Table 3.

Schematic representation of the summary of the results

	BW (17 months)	EI/BW	Insulin sensitivity	Skeletal muscle 2-DG uptake	BMR	EE	PA
IUGR	NC	NC	NC	NC	↓	↓	NC
IPGR	↓	↑	NC	↑ ↑ (SOL, EDL, and GAS)	NC	↑	↑
PNGR	NC	↑	NC	↑ (GAS)	NC	↑	↑

Increase (↑) or decrease (↓) compared with CON. NC, No change; BW, body weight; EI, energy intake; EE, energy expenditure; PA, physical activity.

Discussion

General

The IUGR male rat offspring has previously been examined at various stages of the lifespan and described to be obese with perturbed glucose and insulin concentrations, glucose intolerance, and insulin resistance with associated hyperphagia and physical inactivity (4–8, 12, 13, 32, 33). Regardless of the cause of IUGR, the phenotype of the adult offspring is similar. This is because nutrient restriction in utero was followed by postnatal access to ad libitum milk intake resulting in catch-up growth. The superimposition of catch-up growth resulted in a mismatch between the intrauterine and postnatal nutritional environments, ultimately leading to a chronic disease state (34). The implications of when this phenotype emerges and how it affects the next generation sired by these male offspring are topics of ongoing investigation (35).

In our investigation, the most notable observation was the lack of hyperglycemia, hyperinsulinemia, and insulin resistance in the aging male IUGR offspring. Investigations in younger 10-month-old male IUGR offspring demonstrated a development of sc and visceral adiposity but retention of glucose tolerance and insulin sensitivity (36). The question was whether normal insulin sensitivity encountered in the robust adult male IUGR offspring despite adiposity was: 1) a matter of age, so that aging will display accumulating whole-body insulin resistance and glucose intolerance; or 2) a result of in utero programming from calorie restriction that would be permanently seen throughout life into aging. Hence, it was important to examine the male IUGR adult offspring later in life during aging to resolve this question.

Insulin sensitivity

Intrauterine growth restriction

Previous investigations reporting insulin resistance in the adult male IUGR rat offspring were only based on insulin, glucose, and glucose to insulin ratios (6, 8, 33). Some studies included calculations of the insulin sensitivity index based on glucose and insulin concentrations and surmised the presence of insulin resistance (37, 38).

To overcome the limitations of these previous studies, we conducted hyperinsulinemic-euglycemic clamp experiments. In the presence of exogenous insulin, hyperinsulinemia with normal circulating glucose concentrations, no difference in HGP and glucose R_d was observed in the IUGR vs. CON. Thus, our present observation of retained insulin sensitivity in the aging male IUGR offspring supports a permanently programmed effect due to in utero caloric restriction. This normalcy is achieved by key adaptations, examples being the changes observed in tissue-specific insulin signaling (23, 24) and glucose transporter 4 biology (23).

Previously, other investigators have shown hepatic insulin resistance at 8 wk (12) in the uterine artery-ligated male IUGR offspring. However, the absence of insulin resistance in anesthetized 8-month-old prenatally calorie-restricted male IUGR offspring (22), on the backdrop of insulin resistance seen in 100-d-old anesthetized female IUGR offspring, created some controversy (39). The common approach was hyperinsulinemic-euglycemic clamp but in anesthetized rats. Our present study along with our previous investigation performed at 10 months of age (36) confirms the unequivocal absence of insulin resistance in awake IUGR male offspring in a milieu of adequate or high circulating insulin concentrations.

PNGR and IPGR

Examination of the PNGR and IPGR groups also revealed no changes in basal and insulin-stimulated HGP and R_d. Thus, on the surface, it appeared that superimposition of postnatal caloric restriction on the IUGR offspring failed to have a positive impact particularly during aging. However, when tissue-specific insulin-stimulated glucose uptake was examined, the PNGR demonstrated an increase in the gastrocnemius, whereas the IPGR demonstrated an increase in soleus, extensor digitorum longus, and gastrocnemius muscles vs. that seen in CON or IUGR. This supports a positive impact of IPGR on the IUGR offspring seen as enhanced insulin sensitivity in skeletal muscle. In contrast, no intergroup changes in insulin-stimulated glucose uptake were observed in WAT or liver, the other two insulin-responsive tissues.

Energy metabolism and physical activity

Intrauterine growth restriction

The lack of effect on insulin-induced WAT glucose uptake complements the lack of overt obesity in the aging male IUGR offspring. This may be related to WAT experiencing hypoxic injury and apoptosis during aging (40). However, the energy metabolism particularly in the IUGR reflects a diminution in the basal VO₂ and heat production unlike that seen in the PNGR and IPGR groups. Although fasting with refeeding revealed a similar pattern between the four groups overall, a diurnal variation was evident. VO₂ and heat production were much higher during the dark vs. the light cycle in each group, with the IUGR exhibiting a diminution greater than that of CON. More importantly, the IUGR lacked flexibility in adjusting VO₂ between the fed, fasted, and refed states. In addition, the increase in VO₂ during the dark vs. light cycles was minimal if any in this group.

These perturbations in the energy metabolism are seen in the absence of obesity in the IUGR. However, in the aging IUGR offspring, in the face of no change in energy intake, a reduction in VO₂ and heat production particularly during the light cycle in fed, fasting, and refed states do not reflect physical activity, because no change consistent with inactivity was evident in any cycle. Thus, these changes in the IUGR are reflective of a reduction in the BMR. Significant change in insulin-induced skeletal muscle glucose uptake was associated with no change in skeletal muscle Sirt1, total PKCζ, total AMPK, and pAMPK protein expression. The trend toward an increase of pAMPK in the IUGR vs. CON was suggestive of an energy deficit. The reduction in VO₂, heat production, and BMR are the classical hallmarks of obesity. Hence, it appears that with aging, although overt obesity is not present, features consistent with previous existence of obesity during an earlier age exist.

Other investigations have demonstrated a reduction in physical activity at an earlier adult age in the male IUGR offspring (32), unlike our present observations during aging. It appears that aging results in waning of some of the maladaptive features, such as overt obesity and inactive state, encountered during an earlier adult stage (9–10 months).

PNGR and IPGR

The PNGR and IPGR demonstrated increased energy intake during the two light and dark cycles. The PNGR and IPGR also displayed enhanced VO₂ and physical activity particularly in the dark when compared with CON and IUGR. This change was more acute in the IPGR during fasting and fed rather than the refed states. Further, the increased activity was due to enhanced ambulation and rearing activities. Thus, an argument can be made that in the PNGR and IPGR, particularly during the fed state and in the dark, energy consumption and insulin-stimulated glucose uptake reflect physical activity, because resting energy expenditure was unchanged.

These important observations, particularly in the IPGR, proved to be relatively protective compared with CON when both were placed on a HFD postweaning. Although the CON-HFD were heavier than the IPGR-HFD with more white fat, despite no difference in food (energy) and water intake, enhanced VO₂ and heat production in the IPGR-HFD paralleled the heightened ambulation and rearing activity in this group. Thus, during aging, the impact of postnatal dietary changes modifies the in utero programming effect, influencing the aging process. Early caloric restriction imposed postnatally to match the intrauterine caloric restriction positively affects physical activity and energy balance. All these parameters play a critical role in preventing the adult-type obesity unrelated to in utero programming but encountered when exposed to a postweaning hypercaloric diet. Although augmented insulin-induced skeletal muscle glucose uptake was evident in these two experimental groups, no effect was noted in skeletal muscle Sirt1, total PKCζ, total AMPK, or pAMPK protein concentrations.

Although IPGR had an ameliorating effect on the aging IUGR offspring particularly with respect to glucose and energy metabolism and physical activity further protecting the offspring when exposed to a HFD, it is imperative that future studies ensure the safety of such an early life intervention targeted at preventing the chronic phenotype of the robust and aging adult; for example, postnatal caloric restriction, although retarding growth may result in osteoporosis with aging (41). A significant and beneficial impact of such an early life postnatal intervention on the subsequent metabolic phenotype with aging may also result in unintended consequences on cognition and neuropsychological development.

Summary

In summary, the aging male IUGR offspring was nonobese, not insulin resistant, not hyperphagic but physically active, displaying reduced VO₂, and heat production that were inflexible in response to fasting vs. fed states while still demonstrating minimal diurnal variation. These findings suggest a waning of the overt obesity, inactivity, and dysregulated metabolic phenotype encountered earlier at 10 months of age (36). Specifically, we noted a diminution of energy expenditure reflected by BMR rather than changes in physical activity. In contrast, IPGR similar to PNGR caused a lean, hyperphagic phenotype with enhanced physical activity, which was reflected in increased VO₂ flexible in response to fasting vs. fed states with retained diurnal variation and an enhanced skeletal muscle insulin-stimulated glucose uptake. The enhanced energy expenditure with augmented skeletal muscle insulin sensitivity particularly reflected the state of physical activity in the IPGR unlike that encountered in the IUGR. In addition, IPGR was protective against postweaned consumption of a HFD and its metabolic consequences of obesity (overt and visceral adiposity), energy imbalance, and physical inactivity. Thus, although introduction of postnatal caloric restriction had long-term positive implications reaching into the aging adult, caution must be exercised in balancing the long-term benefits against metabolic dysregulation with the unintended consequences of negatively impacting cognition and neuropsychological state that develops during the early postnatal period. Future investigations should focus on fine tuning postnatal interventions to achieve optimal metabolic and cognitive functions.