Abstract
Objective We aimed to investigate the effects of early high-protein supplementation on low birth weight (LBW)–associated adult metabolic disturbances.
Materials and Methods This study involved 32 LBW rat pups that were fed a normal protein (20% of energy intake) diet or high-protein (30% of energy intake) diet on their first 4 weeks of life. Sixteen rat pups with normal birth weight (NBW) fed the normal-protein diet were included as control. Biochemical measurements were performed at 4 and 12 weeks of age.
Results Low birth weight offspring showed significantly (P < 0.05) increased fat mass percentage and adipocyte size and decreased lean mass percentage and muscle fiber size relative to NBW offspring. These LBW-related changes in body composition were corrected by high-protein diet intervention. At 12 weeks of age, the fasting insulin level (7.14 ± 0.83 vs 9.27 ± 0.67 mU/L) and homeostasis model of insulin resistance (1.71 ± 0.35 vs 2.30 ± 0.44) were significantly lower in high protein–fed LBW offspring than in normal protein–fed LBW offspring. Low birth weight rat pups showed a significant (P < 0.05) reduction in serum adiponectin concentrations, glucose transporter 4 mRNA abundance, and phosphorylation levels of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) relative to NBW controls. These LBW-associated alterations in gene expression were reversed by early high-protein treatment.
Conclusions Early postnatal high-protein intake alters the body composition and improves insulin resistance in adults with LBW, which is associated with activation of the AMPK and mTOR pathways.
Insulin resistance is a major component of the pathogenesis of metabolic diseases including type 2 diabetes, hypertension, and dyslipidemia.1Upon the binding of insulin to its cell surface insulin receptor, glucose transporter type 4 (GLUT4) is translocated from intracellular storage compartments to the plasma membrane, resulting in a rapid increase in glucose uptake. Impaired GLUT4 translocation in skeletal muscle has been linked to development of insulin resistance.2Adiponectin, a collagen-like circulating protein produced and secreted exclusively by adipose tissue, acts as an insulin sensitizer.3Activation of AMP-activated protein kinase (AMPK), a metabolic master enzyme regulating glucose uptake, fatty acid β-oxidation, and GLUT4 biosynthesis, accounts for the insulin-sensitizing effect of adiponectin.4Adiponectin-deficient mice predispose to severe hepatic insulin resistance.5
Low birth weight (LBW) is associated with an increased risk of insulin resistance and type 2 diabetes in adulthood.6It has been suggested that nutritional treatment in early critical developmental ages is able to affect the susceptibility to chronic adulthood diseases, in particular the metabolic disorders, such as insulin resistance, type 2 diabetes, obesity, and hypertension.7Rodríguez-Trejo et al.8reported that protein restriction during pregnancy and/or lactation switches the developmental program of neonatal pancreatic β-cells from proliferation to differentiation, which can contribute to later-life predisposition to type 2 diabetes. These findings reflect “metabolic programming,” a phenomenon whereby a nutritional stress/stimulus applied during critical periods of early development permanently changes an organism’s physiology and metabolism, with consequences observed later in adulthood.9High-protein milk formulas are routinely used to enhance growth in LBW infants. We hypothesized that early high-protein exposure would modify LBW-associated metabolic disturbances in adulthood. To test this, in the present study, high-protein supplementation was given to LBW rat pups during their first 4 weeks of life, and the dietary effects on the growth, body composition, and insulin sensitivity of the animals were examined in later adult life.
MATERIALS AND METHODS
Diets and Animals
A basal diet was prepared based on the American Institute of Nutrition (AIN) purified diet for experimental rodents (AIN-93G),10containing 20% casein as the sole protein source. A high-protein diet containing 30% casein was formulated to be isocaloric by reducing the content of cornstarch. The composition of the 2 experimental diets is shown in Supplementary Table S1 (Supplemental Digital Content 1, http://links.lww.com/JIM/A12).
Specific pathogen-free Sprague Dawley rats aged 13 weeks were purchased from the Medical Animal Experiment Center of Guangdong Province of China and housed in a temperature-controlled room (20-25°C) with a 12-hour light/dark cycle. Male rats were mated with female rats at a ratio of 1 to 2. The day of sperm cell detection in female rats is considered to be day 1 of gestation. Pregnant female rats were randomly assigned to either ad libitum feeding or 40% food restriction from day 1 of gestation. A total of 8 dams were allowed to deliver at term, with birth weight recorded within 12 hours. Low–birth weight rats were defined as having a birth weight more than 2 standard deviations (SDs) below the mean value of newborn rats of dams with ad libitum chow. Sixteen newborns with normal birth weight (NBW) and 32 LBW infant rats were chosen for dietary intervention. The cohort of LBW animals were randomly divided into 2 groups (n = 16 for each group): CL group that was fed the normal protein diet and HPL group that was given the high protein diet. During the suckling period, the rat offspring were reared by foster mothers fed the normal protein diet or the high-protein diet. Pups in the CL group were weaned at 3 weeks of age to ad libitum basal diet, whereas pups in the HPL group were weaned to ad libitum high-protein diet for 1 week followed by the normal protein diet until the end of the experiment at 12 weeks of age. Normal birth weight rats (CN group) received the same intervention with the CL group. Body weights were recorded weekly. Eight animals from each group were euthanized with an overdose of 10% chloral hydrate administered intraperitoneally at 4 and 12 weeks and subjected to further analyses as described below. The protocol of this study was depicted in Figure 1 and approved by the institutional animal care and use committee of Medical Animal Experiment Center of Guangdong Province of China.
Body Composition Analysis
Measurement of whole body composition was performed according to previous studies11using a dual-energy X-ray absorptiometry scanner (Hologic QDR-4500A; Hologic, Waltham, MA). Each animal was placed in a prone position. Several parameters were obtained: whole body weight, fat mass, fat mass percentage, lean mass, lean mass percentage, bone mineral content, and bone mineral content percentage.
Histomorphometric Analysis
Histomorphometric analysis was conducted as previously described.12Samples of visceral adipose tissue and biceps femoris muscle tissue from experimental animals were fixed in 10% formalin, embedded in paraffin, and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO). Photomicrographs were taken by a light microscope (model BX60; Olympus Ltd., Tokyo, Japan) and analyzed with a computer-assisted system (Image-Pro Plus 5.1; Media Cybernetics, MA). Ten random high-power fields (magnification, ×400) were evaluated for each section, and the cross-sectional area of adipocytes and muscle fibers was determined.
Measurement of Blood Glucose, Insulin, and Adiponectin
Orbital vein blood samples were collected after a 12-hour fasting, and serum was immediately separated for further analyses. Serum glucose was measured using the Blood Glucose Test Kit (Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Serum insulin and adiponectin levels were measured using the Insulin Radioimmunoassay Kit (Beijing Atom HighTech Co., Ltd., Beijing, China) and using the rat adiponectin enzyme-linked immunosorbent assay kit (Millipore, Bedford, MA), respectively. The blood sample (0.5-1 μL) was used for each assay. The homeostasis model assessment-insulin resistance (HOMA-IR) index was calculated as [fasting serum glucose × fasting serum insulin/22.5], with lower values indicating a higher degree of insulin sensitivity.13
Western Blot Analysis
Muscle tissues from rats were homogenized and lysed in a lysis buffer containing a protease inhibitor phenylmethylsulfonyl fluoride (1 mM; Beyotime Institute of Biotechnology, Jiangsu, China). Protein concentrations were determined using the bicinchoninic acid method (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Samples of protein extracts were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% bovine serum albumin (Sigma-Aldrich), the membranes were incubated overnight at 4°C with primary antibodies against mammalian target of rapamycin (mTOR; #2983), phosphorylated mTOR (Ser2448) (p-mTOR; #2971), AMPKα (#2603), and phosphorylated AMPKα (Thr172) (p-AMPKα; #2535; Cell Signaling Technology, Beverley, MA), followed by incubation with appropriate horseradish peroxidase-linked secondary antibodies (Cell Signaling Technology). Bound antibodies were visualized by the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE). Signal intensities were densitometrically quantitated using the Quantity One Software (Bio-Rad, Hercules, CA). The signals from each of the phospho-specific immunoblots were normalized against the total protein amounts.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA from muscle tissues were extracted with TRIzol, following the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Reverse transcription was performed with random primers (Invitrogen) and M-MLV reverse transcriptase (Invitrogen). Real-time PCR amplification was conducted on the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) using SYBR Green PCR Master Mix (Life Technologies Corporation, Foster City, CA). The PCR primers for GLUT4 (Cat. No. RQP049078) and for GAPDH (Cat. No. RQP049537) were purchased from GeneCopoeia (Germantown, MD). All assays were performed in triplicate, and the threshold cycle (Ct) was calculated. The relative mRNA expression level normalized by GAPDH mRNA level was then determined using the 2–ΔΔCt method.14
Statistics
Using two-sided 5% significance level, a sample size of 8 animals per group was needed. This gave an 80% power to detect a 14% difference in fasting serum insulin levels between treatments. All quantitative data were expressed as mean ± SD and analyzed using 1-way analysis of variance followed by the least significant difference post hoc test. P values less than 0.05 were considered as statistically significant. Statistical differences were calculated using the SPSS 16.0 (SPSS Inc., Chicago, IL).
RESULTS
Body Weight and Composition
Birth weight was significantly reduced in the offspring from food-restricted dams as compared with the offspring from control pregnant dams (5.03 ± 0.28 vs 7.05 ± 0.48 g; P < 0.05). At 4 weeks of age, high-protein diet–treated LBW offspring had a significantly higher weight gain in comparison with basal diet–treated LBW offspring (86.07 ± 5.19 vs 77.96 ± 3.49 g; P < 0.05; Table 1). However, at 12 weeks of age, body weight was similar in all the groups tested. High-protein intervention resulted in an increase in the lean mass percentage (83.36 ± 1.73% vs 78.82 ± 2.13%) and a reduction in the fat mass percentage (14.07 ± 1.75% vs 18.58 ± 2.15%) as compared with the CL group (Table 1). However, there was no statistical difference in the bone mineral content percentage between the 2 groups. Animals in both the CN and HPL groups had similar body weights and compositions at 4 and 12 weeks of age.
High-Protein Diet Reduces Adipocyte Size and Increases the Size of Muscle Fibers
Histomorphometric analysis of visceral fat tissue revealed that adipocyte size was significantly lower in high-protein diet–treated LBW offspring compared with the CL group (5548 ± 578 vs 6852 ± 832 μm2; P < 0.05; Fig. 2A, Table 2). By contrast, the cross-sectional area of skeletal muscle fibers was significantly greater in the HPL group compared with the CL group (3176 ± 300 vs 2612 ± 296 μm2; P < 0.05; Fig. 2B, Table 2). No statistical difference in the size of adipocytes or muscle fibers was observed between the HPL and CN groups.
Improvement of LBW-Associated Insulin Resistance by High-Protein Diet
At 4 weeks of age, all the groups studied had similar fasting serum glucose and insulin levels and HOMA-IR indices (Table 3). At 12 weeks of age, the HOMA-IR index (1.71 ± 0.35 vs 2.30 ± 0.44) and fasting insulin level (7.14 ± 0.83 vs 9.27 ± 0.67 mU/L) were significantly (P < 0.05) decreased in the HPL group relative to the CL group. In contrast to HOMA-IR and fasting serum insulin, no significant changes in fasting serum glucose were observed. As shown in Figure 3A, serum adiponectin levels were profoundly lower in the CL group than in the CN or HPL group (P < 0.05). Moreover, animals in the CL group showed a significantly reduced amount of skeletal muscle GLUT4 mRNA as compared with the CN or HPL group (P < 0.05; Fig. 3B). These results suggest that high-protein diet contributes to an improvement of insulin resistance associated with LBW.
High-Protein Diet Promotes the Phosphorylation of mTOR and AMPKα
Western blot analysis of skeletal muscle tissues revealed that there was a significantly lower amount of phosphorylated mTOR and AMPKα in the CL group in comparison with the CN or HPL group (P < 0.05; Fig. 4). However, the total mTOR and AMPKα levels remained unchanged among the groups studied.
DISCUSSION
In this study, we showed that high-protein intervention significantly altered the body composition of LBW rat offspring, with an increased percentage of lean mass and decreased percentage of fat mass. The offspring with LBW were predisposed to insulin resistance manifested as higher HOMA-IR indices and fasting insulin levels. Notably, the HOMA-IR index and fasting insulin level were significantly lowered in the LBW offspring fed by high-protein diet. These results highlight the benefits of high-protein intervention in modulating the body composition and insulin response of the LBW offspring.
Low birth weight indicates an elevated risk for metabolic syndrome in young adults. Fagerberg et al.15reported that LBW in combination with catch-up growth is closely correlated with the occurrence of the metabolic syndrome in the late middle age. In line with these reports, we found that LBW rats fed the basal diet developed insulin resistance at 12 weeks of age and had similar body weight to NBW controls fed the same diet. It has been established that there is a link between birth weight and body composition. Ylihärsilä et al.16reported that LBW is associated with lower lean mass in adult life. Consistently, we found that the LBW offspring showed a decrease in the lean mass percentage and a concomitant increase in the fat mass percentage compared with NBW controls. Histomorphometric analysis further revealed that the LBW offspring had a significantly higher cross-sectional fat area and lower cross-sectional muscle area than NBW controls. Using a mouse model of LBW, Isganaitis et al.17demonstrated that fat mass accrues preferentially during catch-up growth in LBW newborns and LBW adults have increased adiposity, which is primarily because of upregulation of the lipogenic genes Fasn, AccI, Lpin1, and Srebf1. Increasing adipogenesis may represent another programmed response to early postnatal catch-up growth. It has been reported that postnatal catch-up growth after fetal malnutrition programs proliferation of preadipocytes in rats.18It is therefore of interest to check whether LBW offspring show an enhanced adipogenic response during catch-up fat deposition. Excessive fat accumulation during childhood has deleterious effects and is associated with reduced insulin sensitivity in LBW adults.19These results collectively suggest that the catch-up growth in LBW offspring results in increased fat deposition, which may in turn contributes to the development of insulin resistance later in life. High-protein intervention can prevent the preferential accumulation of the catch-up fat and, thus, have beneficial effects on insulin response. Indeed, LBW-associated insulin insensitivity was profoundly improved by high-protein treatment.
Adipose tissue-derived cytokines (adipokines), such as adiponectin, leptin, resistin, and visfatin, have been suggested to link adiposity to insulin resistance.20Extensive studies have indicated that adiponectin has insulin-sensitizing properties.3We found that high-protein diet resulted in a marked increase in serum adiponectin levels, which may partially explain improved insulin sensitivity in LBW newborns fed high-protein diet. In adult rats, high-protein treatment also resulted in greater serum adiponectin levels than normal-protein diet.21It is well accepted that reduced GLUT4 in the skeletal muscle contributes to insulin resistance.22Adiponectin has been reported to be able to increase GLUT4 translocation and glucose uptake in rat skeletal muscle cells.23In agreement with increased adiponectin levels, high-protein supplementation induced a profound elevation in the abundance of skeletal muscle GLUT4 mRNA.
The mTOR acts as a central regulator of protein synthesis and is associated skeletal muscle hypertrophy. Chotechuang et al.24showed that in rats fed a high protein diet for 14 days, the hepatic p-mTOR was higher compared with that in rats receiving a normal protein diet. We also found that the phosphorylation level of mTOR in the skeletal muscle was significantly elevated in the LBW offspring fed high protein diet in comparison with those fed basal diet, which provides an explanation for the increased percentage of lean mass and larger muscle size in the HPL group. It is well established that activation of the AMPK signaling improves insulin responsiveness. Watt et al.25showed that activation of skeletal muscle AMPK is responsible for the reversal of obesity-induced insulin resistance by ciliary neurotrophic factor. It has been described that epigallocatechin-3-O-gallate attenuates free fatty acid–induced peripheral insulin resistance through the AMPK pathway.26Zhang et al.27reported that myostatin-null mice exhibits reduced fat accumulation and peripheral insulin resistance when compared with wild-type mice, which is due to activation of the AMPK pathway. In line with these studies, we noted that the improvement of insulin sensitivity by high-protein diet was accompanied by enhanced phosphorylation of AMPKα, indicative of activation of the AMPK pathway. Activation of AMPK can facilitate GLUT4 translocation and, thus, reduce insulin resistance.28Indeed, the skeletal muscle GLUT4 mRNA level was increased in accord with the AMPKα phosphorylation in high-protein diet–treated LBW rats. In contrast to AMPK, activation of the mTOR signaling, at least under some conditions, leads to insulin resistance.29Several lines of evidence have indicated an interaction between the AMPK and mTOR signaling pathways.30It remains to be addressed whether the consequence of the mTOR signaling on insulin responsiveness could be influenced by the AMPK signaling. Additionally, in this study, we included both male and female SD rat pups, without considering the potential sexual effects. However, it is interesting to determine whether there are sex-specific differences in the outcomes of high-protein diet intervention in LBW offspring.
In summary, our data demonstrate that early high-protein supplementation alters the body composition and improves insulin sensitivity in rat offspring with LBW, which is coupled with an increase in serum adiponectin levels and skeletal muscle GLUT4 mRNA abundance. These effects of high-protein diet may be associated with activation of the AMPK and mTOR signaling pathways.