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The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 51-56
USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030-2600
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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We tested the hypothesis that chronic ingestion of increased concentrations of milk-borne des(1-3) human insulin-like growth factor-I (hIGF-I) stimulates gastrointestinal growth and development in suckling mice. We used a transgenic mouse with targeted, lactation-dependent, overexpression of des(1-3) hIGF-I in the mammary gland (IGF). Pups were suckled (7 pups per litter) from birth by either IGF (n = 3-6 litters) or control (n = 3-5 litters) dams. In IGF and control pups, we measured the growth (protein and DNA content) and protein synthesis rate (3H-phenylalanine incorporation) of gastrointestinal and visceral organs in 4-, 8-, 12-, 16- and 29-d-old pups. Des(1-3) hIGF-I in milk from IGF dams was 40-200-fold higher than mouse IGF in either IGF or control dams, but was not detected in the plasma of pups suckling IGF dams. Small intestinal weight, protein and DNA content at 8 and 16 d were greater in pups suckling IGF dams than control dams; protein synthesis was also greater in IGF pups at 8 d. Total intestinal lactase activity at 8 and 12 d of age tended to be higher (P < 0.10) in IGF than in control pups. Hypersecretion of des(1-3) hIGF-I in milk ingested by suckling mice pups had limited effects on the growth and maturation of the gastrointestinal tract. Moreover, there was little evidence that milk-borne IGF-I is absorbed into the circulation and stimulates visceral organ growth. This study also demonstrates the feasibility of using mammary-specific transgenes to increase the concentration of milk-borne growth factors to examine whether they affect the growth and development of the suckling neonate.
KEY WORDS: growth factors · lactase · mice · neonatal · protein synthesis
Insulin-like growth factor-I (IGF-I)4 and the truncated form, des(1-3)IGF-I, have been identified in mammary secretions of several species (Donovan et al. 1991 Several studies have shown that systemic administration of recombinant human IGF-I and its related analogs has trophic effects on intestinal tissues under a wide variety of physiologic conditions (Lemmey et al. 1991 In this study, we used a recently developed transgenic mouse (Hadsell et al. 1996 Transgenic mice.
The whey-acidic protein IGF-I (WAP-IGF) transgene was constructed using a polymerase chain reaction-based strategy to replace exons 1-4 of the rat whey acid protein gene with human IGF-I coding sequences (Hadsell et al. 1996 Animals and design.
We studied multiple litters of pups suckled by either WAP-IGF transgenic (IGF) or control dams (Control); control dams were from a nontransgenic crossbred strain (ICR × B6C3F1). When pups were 1 d old, litter size was equalized to seven pups to minimize any confounding effects of variation in pup milk intake (Fiorotto et al. 1991 In vivo protein synthesis and tissue assays.
In each litter, we measured protein synthesis in vivo in three randomly selected pups using a modification of the technique described by Garlick et al. (1980) Assay of human-specific and mouse-specific IGF-I in plasma and milk.
Maternal defatted milk (150 µL) and neonatal pup plasma (30-50 µL) were acid-ethanol extracted and assayed for human IGF-I concentration using a human-specific two-site immunoradiometric assay that recognizes both full-length IGF-I and des(1-3)IGF-I peptides (Diagnostic Systems Laboratories, Webster, TX). The sensitivity of the immunoradiometric human IGF-I assay used was ~2 pg per tube. The inter- and intra-assay variation was 9.8 and 7.9%, respectively. The concentration of mouse IGF-I in the extracts of milk and plasma was measured using a RIA specific for rodents (Diagnostic Systems Laboratories, Webster, TX). All of the samples were analyzed in one assay with a coefficient of variation of 8.4%. The protein concentration in the milk supernatant after centrifugation at 10,000 × g for 10 min was determined as described by Smith et al. (1985) Statistics.
Data were analyzed by two-way ANOVA using litter as the experimental unit and dam genotype and postnatal age as the main effects. Significant genotype effects at a given age were analyzed by one-way ANOVA. When postnatal age was significant, differences between ages were analyzed using Fisher's multiple comparisons test. Results are presented as means ± SD. A probability value <0.05 was considered significant.
The average pup weight at 4, 8, 12, 16 and 29 d of age did not differ between IGF and control litters (Table 1). The stomach contents in IGF and control pups were similar at all ages studied (data not shown). The weight (Table 1) and protein content (data not shown) of the stomach at 12 d were significantly higher in pups suckled by IGF compared with control dams. The relative weights of the liver, spleen and kidney were not different between IGF-I and control pups (Table 1). In general, the protein and DNA contents of the liver and spleen were similar at all ages in both IGF and control pups; however, the spleen protein content at 16 d was lower in IGF than in control pups (data not shown). The stomach protein content at 16 d was lower in IGF than in control pups. The relative weight and protein content of the small intestine at 8 and 16 d were significantly higher in pups suckled by IGF compared with control dams (Table 2). The small intestinal DNA content at 16 d was higher in IGF than in control pups.
There has been considerable interest in the function of milk-borne growth factors since Klagsburn's original finding that human milk stimulates the proliferation of cultured fibroblasts (Klagsburn 1978
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Francis et al. 1986, Simmen et al. 1988). The des(1-3)IGF-I variant is more potent than native IGF-I because of its reduced binding affinity for the IGF binding proteins (IGFBP). Studies have demonstrated that IGF-I acts as a progression factor during the G1-phase of the cell cycle (Baserga and Rubin 1993), activates cellular protein synthesis (Ballard et al. 1986, Francis et al. 1986) and inhibits apoptosis or programmed cell death (Harrington et al. 1994). The intestinal mucosa contains a multitude of cell types that have especially high rates of protein synthesis (Burrin et al. 1991, McNurlan et al. 1979) and undergo continuous proliferation and apoptosis (O'Connor 1966, Potten et al. 1995). However, the extent to which milk-borne IGF-I modulates these cellular functions in the gastrointestinal tract of the suckling neonate has not been established.
, Read et al. 1992, Steeb et al. 1994, Zhang et al. 1995). Many of these studies have shown that des(1-3)IGF-I is a more potent stimulus of intestinal growth than the full-length IGF-I peptide, suggesting that the IGF-binding proteins mitigate the anabolic action of IGF-I in the intestine. These and other studies indicate that systemic IGF-I increases intestinal crypt cell proliferation, protein synthesis, mucosal thickness and length (Lo and Ney 1996, Potten et al. 1995). Although few studies have examined the trophic effect of orally administered IGF-I, some recent studies with neonatal animals (Baumrucker et al. 1994, Houle et al. 1997b, Philipps et al. 1995, Xu et al. 1994) have shown that oral administration of physiologic doses of rhIGF-I in formula results in measurable increases in intestinal crypt cell proliferation and lactase activity, but no demonstrable increase in intestinal mucosal mass or length. In a recent study using neonatal pigs (Burrin et al. 1996), we demonstrated that oral administration of a supraphysiologic dose of rhIGF-I in formula increased small intestinal mucosal growth and was associated with increased villous height. Because the previous studies used artificial formulas and feeding conditions, we wished to determine whether providing increased concentrations of IGF-I, secreted naturally in maternal milk, would affect intestinal growth in normal, suckling neonatal mice. The model we used administered the more biologically potent des(1-3)IGF-I peptide to the suckling neonate, which eliminates any potential inhibitory actions of IGFBP present in maternal milk (Donovan et al. 1991, Hadsell et al. 1996) and is secreted locally by the intestinal mucosal cells (Park et al. 1992, Simmons et al. 1995).
), which exhibits targeted overexpression of des(1-3) human IGF-I in the mammary gland and milk. We tested the following two hypotheses: 1) increased concentrations of des(1-3) human IGF-I, when ingested naturally in mother's milk, results in increased gastrointestinal growth in suckling neonatal offspring; and 2) maternal des(1-3) human IGF-I ingested by suckling pups is absorbed into the circulation and stimulates the growth of other visceral organs.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Selective deletion of a 9-bp sequence from a portion of the IGF-Ia fragment in the original WAP-IGF minigene construct resulted in plasmids designed to target des(1-3)hIGF-I to the mammary gland. Generation, characterization and genotyping of the transgenic mice have been described previously (Hadsell et al. 1996). The WAP-IGF-I transgenic mice used in these studies were from line 8266 derived from a crossbred strain (ICR × B6C3F1).
). Pups were allowed to suckle normally on their dams until the day of study. On that day, pups were removed from their dam and deprived of food for 2 h to allow for gastric emptying in the pups and accumulation of milk in the dam's mammary glands. The pups then were returned to their dam and allowed to suckle for 2 h at which time we measured protein synthesis in vivo. This protocol was designed to minimize any variation in milk intake between pups and to ensure that all pups were studied in the fully fed state. About 2-3 h after terminal removal of all pups, dams were lightly anesthetized and milk samples were collected from multiple mammary glands as described previously (Fiorotto et al. 1991). At 4, 8, 12, 16 and 29 d of age, three pups each from either IGF or control litters were studied. Pups studied at 29 d of age were weaned from their respective dam at 21 d of age and fed a nonpurified diet (Purina Rodent diet, Purina Mill, St. Louis, MO) until d 29. At 4, 12, 16 and 29 d, we studied litters (n = 3) from each genotype (i.e., control or IGF); at 8 d we studied either six (IGF) or five (control) litters from each genotype. The protocols were approved by the Animal Care and Use Committee of Baylor College of Medicine, and were conducted in accordance with NIH guidelines (NRC 1985).
. Pups were given an intraperitoneal injection of L-[4-3H]-phenylalanine [Amersham Life Sciences, Arlington Heights, IL, 4-5 MBq per pup, 150 µmol Phe/100 g body weight (BW)] in sterile water and killed by decapitation 15 min later. The peritoneal cavity was immediately flushed with ice-cold 9 g/L saline to remove any unabsorbed radioisotope and chill the tissues. The gastrointestinal tract, liver, spleen and kidneys were rapidly removed, weighed and frozen in liquid nitrogen. The small intestine tissue was separated from the stomach and large intestine and flushed with ice-cold saline before being weighed. The large intestine weight was not measured. The stomach contents were removed and weighed before the stomach tissue was frozen. All tissue dissections were performed at 4°C.
). Protein synthesis was calculated as a fractional rate (Ks, %/d) from the equation described by Garlick et al. (1980) as follows:
where Sb is the specific activity of the perchloric acid (PCA)-insoluble or protein-bound phenylalanine pool (dpm/nmol), Sa is the specific activity of the PCA-soluble or tissue free phenylalanine pool (dpm/nmol), and t is time of labeling in minutes. Tissue protein and DNA content were determined as described by Smith et al. (1985)
and Labarca and Piagen (1980), respectively. Intestinal lactase and sucrase activities were determined in tissue homogenates as described by Dudley et al. (1994). The total intestinal activity [µmol/(min · g BW)] of lactase and sucrase was calculated as the product of the specific activity [µmol/(min · g protein)] and the total small intestinal protein content (µg/g BW).
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RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Body weights and relative organ weights of mouse pups suckled by wild-type (Control) and des(1-3)hIGF transgenic (IGF) dams1
View this table:
Table 2.
Weight, protein and DNA content of the small intestine of mouse pups suckled by wild-type (Control)
and des(1-3)hIGF transgenic (IGF) dams1
View larger version (49K):
[in a new window]
Fig 1.
Fractional protein synthesis rates (%/d) of stomach (upper panel) and small intestine (lower panel) of mouse pups suckled by wild-type (control) and des(1-3) human insulin-like growth factor-I (IGF-I) transgenic dams. Bars represent means ± SD, n = 3-6 litters. *Control and IGF groups differ, P < 0.05.
View larger version (44K):
[in a new window]
Fig 2.
Fractional protein synthesis rates (%/d) of liver (upper panel) and spleen (lower panel) of mouse pups suckled by wild-type (control) and des(1-3) human insulin-like growth factor I (IGF-I) transgenic dams. Bars represent means ± SD, n = 3-6 litters. *Control and des(1-3) insulin-like growth factor-I (IGF) groups differ, P< 0.05.
View this table:
Table 3.
Intestinal lactase and sucrase activities in mouse pups suckled by wild-type (Control) and des(1-3)hIGF transgenic (IGF) dams1
) using the same immunoradiometric assay. The immunoradiometric assay used has been shown to be nonreactive to both mouse and rat IGF-I and is specific for hIGF-I, but does not distinguish between the full-length and truncated des(1-3)hIGF-I forms. Despite the difference in des(1-3) hIGF-I content, however, the protein concentrations in milk did not differ between IGF and control dams. Human IGF-I was not detected in plasma samples measured 2 h after feeding in any of the IGF pups studied, regardless of age. The concentration of human IGF-I in the milk from IGF dams ranged from ~40- to 200-fold higher than that of mouse IGF-I. The concentration of human IGF-I in milk from IGF dams was approximately four- to fivefold higher at 8 and 16 d than at 4 and 12 d. The concentration of des(1-3) hIGF-I in milk from IGF dams was poorly correlated (r2 = 0.05; P = 0.41) with the intestinal weight of the respective pups, across all ages studied. The concentration of mouse IGF-I in milk at 4 and 8 d was significantly lower in IGF than in control dams. There were no differences in the protein concentration in milk collected from IGF and control dams at any age; the mean (± SD) milk protein concentrations (g/L) among control and IGF dams were 82 ± 14, 124 ± 22, 101 ± 6 and 139 ± 23 at 4, 8, 12 and 16 d of pup age, respectively. The plasma concentrations of mouse IGF-I were not significantly different in IGF and control pups at any age studied. However, the plasma concentration of mouse IGF-I in both groups increased (P < 0.05) between 4 and 29 d of age.
View this table:
Table 4.
Human and rodent-specific IGF-I concentrations in plasma of mouse pups and in milk from wild-type (Control)
and des(1-3)hIGF transgenic (IGF) dams1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). It has been hypothesized that the IGF-I found in milk plays a functional role in the growth and development of the neonate, particularly the gastrointestinal tract (Burrin 1997, Donovan and Odle 1994). In this study, we used a novel transgenic approach (Hadsell et al. 1996) to determine whether providing increased concentrations of des(1-3)IGF-I, naturally secreted in maternal milk, would affect gastrointestinal growth in normally suckling neonatal mice. The main advantage of this approach is that it allowed us to manipulate the concentration of IGF-I in milk without the use of artificial formulas and invasive feeding protocols.
, Philipps et al. 1997). However, the magnitude of the trophic response was much less than that observed in neonatal pigs and was not consistent at all ages. The stimulation of intestinal mucosal growth occurred by hypertrophy (8 and 16 d) and hyperplasia (16 d), based on the protein and DNA contents. Previous studies have demonstrated that IGF-I stimulates growth via both protein synthesis and cell proliferation (Lo and Ney 1996, Park et al. 1992, Potten et al. 1995). The greater protein content at d 8 likely resulted from increased protein synthesis; however, we found no evidence for increased cell proliferation.
, Ma and Xu 1997, Young et al. 1990). We found that total lactase activity tended (P < 0.10) to be higher in IGF than in control pups at 8 and 12 d of age. However, the increased lactase activity at 8 d resulted from increased intestinal mass, whereas the increase at 12 d resulted from an increase in specific activity. The greater total sucrase activity in IGF pups at 16 d was largely a result of greater intestinal mass. The mechanism by which oral IGF-I increases intestinal lactase has not been established. However, a recent study in neonatal pigs suggests that oral IGF-I may increase the synthesis and post-translational processing of prolactase (Houle et al. 1997a).
, Burrin et al. 1996, Houle et al. 1997b) and that the intestinal absorption of orally administered 125I-IGF-I is minimal (Donovan et al. 1997, Philipps et al. 1995). Furthermore, with the exception of protein synthesis in the spleen, the absence of any significant increase in body weight or visceral organ weight in pups suckled by IGF-I dams also suggests that the milk-borne des(1-3)IGF-I had only a localized effect in the small intestine. It is difficult to ascribe the increased spleen protein synthesis to a direct effect of milk-borne des(1-3)IGF-I because we did not detect it in the plasma of pups suckled by IGF dams. However, given the sensitivity of the spleen to des(1-3)IGF-I, we cannot exclude the possibility that some limited intestinal absorption of milk-borne des(1-3)IGF-I may have increased spleen protein synthesis.
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FOOTNOTES |
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Manuscript received 8 July 1998. Initial reviews completed 31 July 1998. Revision accepted 30 September 1998.
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ACKNOWLEDGMENTS |
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The authors gratefully acknowledge the technical assistance of Xiaoyan Chang, Susan McAvoy, Shahed Ziari and Elizabeth Hopkins. We also thank Leslie Loddeke for editorial assistance, Adam Gillum for graphics and Jane Schoppe for secretarial assistance in the preparation of this manuscript.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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