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Articles

Oral IGF-I Alters the Posttranslational Processing but Not the Activity of Lactase-Phlorizin Hydrolase in Formula-Fed Neonatal Pigs^{1 ,2}

Douglas G. Burrin^*3, Barbara Stoll^*, Ming Z. Fan^*,4, Mary A. Dudley, Sharon M. Donovan and Peter J. Reeds^**

^* U. S. Department of Agriculture Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030; Division of Physiology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103; and Departments of ^** Animal Sciences and Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801

³To whom correspondence should be addressed. E-mail: dburrin{at}bcm.tmc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To determine the cellular mechanism whereby oral insulin-like growth factor I (IGF-I) increases intestinal lactase-phlorizin hydrolase (LPH) activity, we studied 2-d-old pigs fed cow’s milk formula (control, n = 5), formula + low IGF-I (0.5 mg/L; n = 6) or formula + high IGF-I (12.0 mg/L, n = 6) for 15 d. On d 15, intestinal protein synthesis and lactase processing were measured in vivo in fed pigs using a 6-h intravenous, overlapping infusion of multiple stable isotopes (²H₃-Leu,¹³C₁-Leu,¹³C₁-Phe,²H₅-Phe,¹³C₆-Phe and ¹³C₉-Phe). Morphometry and cell proliferation also were measured in the jejunum and ileum. Neither dose of IGF-I affected the masses of wet tissue, protein or DNA, or the villus height, cell proliferation or LPH-specific activity. Oral IGF-I decreased the synthesis and abundance of prolactase-phlorizin hydrolase (pro-LPH), but increased brush-border (BB)-LPH synthesis in the ileum. The BB-LPH processing efficiency was twofold to threefold greater in IGF-fed than in control pigs. In all pigs, villus height and the total mucosal and specific activity of LPH activity were greater in the ileum than in the jejunum, yet the synthesis of BB-LPH were significantly lower in the ileum than in the jejunum. We conclude that oral IGF-I increases the processing efficiency of pro-LPH to BB-LPH but does not affect LPH activity. Moreover, the posttranslational processing of BB-LPH is markedly lower in the ileum than in the jejunum.

KEY WORDS: • protein degradation • cell proliferation • growth factor • protein synthesis • disaccharidase • pigs

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Lactase-phlorizin hydrolase (LPH)⁵ is the major intestinal disaccharidase responsible for the hydrolysis of the lactose in milk and is thus, an important factor in energy utilization in developing mammals. The expression of LPH activity is developmentally programmed and reaches a maximum late in gestation, presumably to prepare the newborn for digestion of milk lactose (1 ,2) . Consistent with the ontogeny of LPH expression, studies in human infants have shown that LPH activity and lactose digestion are positively correlated with gestational age and may be incomplete or deficient in premature infants (3 4 5) . The limited lactose digestive capacity in premature infants has been linked to feeding intolerance and increased incidence of necrotizing enterocolitis (6 ,7) .

In the developing neonatal intestine, several factors have been shown to affect the expression and activity of LPH, including diet (5 ,8 9 10) , the proximal-distal location (11 ,12) and stage of enterocyte differentiation along the crypt-villus axis (13) . At the cellular level, the production of LPH is subject to multiple sites of regulation at the transcriptional, translational and posttranslational levels. In pigs, at least two isoforms of prolactase-phlorizin hydrolase (pro-LPH), one mannosylated and the other bearing complex glycosidic side chains, have been isolated from the small intestinal mucosa. Furthermore, the complex glycosylated precursor form is cleaved proteolytically before insertion into the brush-border (BB) membrane as BB-LPH. Thus, in vivo, a long time can elapse between translation of pro-LPH and final insertion, and a number of studies (9 ,14) have indicated that <100% of pro-LPH eventually appears in the BB membrane. Our studies in newborn pigs have shown that feeding colostrum stimulates the synthesis of pro-LPH, but its posttranslational processing to BB-LPH form is disrupted (8 ,9) . This observation prompted us to examine the factors in colostrum that may increase the synthesis and activity of lactase in the neonate.

Insulin-like growth factor I (IGF-I) is abundant in colostrum (15) , and a number of recent studies with neonatal pigs and rodents have demonstrated that orally administered IGF-I stimulates intestinal growth (16 17 18) , lactase activity (19 20 21 22) and glucose transport activity (23) . In contrast, some studies with neonatal rodents have shown very limited effects of oral IGF-I on either intestinal growth or lactase activity (24 ,25) . In neonatal pigs, the effect of oral IGF-I on intestinal growth and lactase activity is dose-dependent, in that intestinal growth seems to be stimulated only at pharmacological doses (16) , whereas lactase activity responds to a physiological dose of the peptide (19) . A subsequent study demonstrated that the increased lactase activity was associated with increased abundance of pro-LPH and LPH mRNA, implying that oral IGF-I increases lactase gene expression (24) . However, the effects of IGF-I on pro-LPH synthesis and posttranslational processing have not been measured. Therefore, the main objective of the current study was to quantify the effects of IGF-I supplemented in formula at two doses on pro-LPH synthesis and the efficiency of its transfer to the BB membrane. We measured the synthesis rate of pro-LPH and the proportion transferred to BB-LPH in neonatal pigs, using a recently developed multiple stable isotopic approach (15) that allows detailed kinetic analysis in a single tissue sample. In addition, we assessed whether the potential effects of oral IGF-I on LPH synthesis and activity were associated with changes in cell proliferation rate and villus morphometry.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals, diets and design.

Pregnant sows were purchased from the Texas Department of Criminal Justice (Huntsville, TX). The sows were fed a commercial nonpurified diet (Purina Mills, St. Louis, MO). Pregnant sows were housed in the Children’s Nutrition Research Center (Houston, TX) at 24°C in free-standing farrowing crates; they consumed food and water ad libitum. Pigs were delivered vaginally between 113 and 114 d of gestation and allowed to suckle the dam naturally to obtain colostrum for 24–36 h. They were then weaned completely from their dams and randomly assigned to receive either a cow’s milk formula (Litterlife; Merrick, Uniondale, WI; control group) or formula supplemented with a low (0.5 mg IGF-I/L; low IGF) or high (12.0 mg IGF-I/L; high IGF) dose of human recombinant IGF-I (Genentech, San Francisco, CA) for 14 d. Pigs were housed individually in stainless steel cages in a room maintained at 29–30°C and fed 300–350 mL of formula per kg body daily, using an artificial feeding device similar to that described previously (19) . Briefly, a fluid reservoir was connected with tubing to an infant bottle nipple that was attached to the side of the cage. The piglet obtained liquid formula by sucking on the nipple. The reservoir was filled four times daily to ensure that formula was constantly available. The formula was prepared daily and mixed with water at the ratio of 120–150 g powder/L. The dry matter content of the formula was gradually increased in the first 7 d of feeding to minimize diarrhea. The nutrient content of the formula ranged from 30 to 37.5 g protein/L and from 500 to 630 kcal (2.09–2.63 MJ) gross energy/L. After 9 d of dietary treatment, all pigs were deprived of food overnight. The following morning, a polyvinyl chloride catheter (1.78 mm o.d.) was surgically implanted in the carotid artery and external jugular vein of the pigs under general anesthesia. The pigs were ambulatory within 4–6 h and resumed their presurgical rates of formula intake within 24 h after surgery. A total of 17 pigs from three litters was studied, and within each litter all three treatment groups were replicated (control, n = 5; low IGF, n = 6; high IGF, n = 6). The protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Stable isotope infusion protocol.

After 14 d of treatment, pigs were given a multiple overlapping infusion of stable isotopes similar to that described previously (15) . Pigs were infused intravenously with a total of six stable amino acid isotopomers, and the total infusion time was 360 min. All isotopes were the L-form of the amino acid and were purchased from Cambridge Isotope Laboratories (Andover, MA). Two stable isotopomers of leucine were infused at the following rates: [5,5,5-²H]-leucine (²H₃-Leu) at 30 µmol · kg^-1 · h^-1 for 360 min; [1-¹³C]-leucine (¹³C₁-Leu) at 30 µmol · kg^-1 · h^-1 for 240 min. Four stable isotopomers of phenylalanine were infused at the following rates: [1-¹³C]-phenylalanine (¹³C₁-Phe) at 20 µmol · kg^-1 · h^-1 for 180 min; [ring D₅-²H]-phenylalanine (²H₅-Phe) at 20 µmol · kg^-1 · h^-1 for 90 min; [ring -¹³C₆]-phenylalanine (¹³C₆-Phe) at 20 µmol · kg^-1 · h^-1 for 60 min; U-[¹³C]-phenylalanine (U-¹³C₉-Phe) at 30 µmol · kg^-1 · h^-1 for 30 min. During the infusion protocol, pigs were fed by gavage half of the total daily intake (150 mL/kg) of their respective dietary treatments as follows: 75 mL/kg at time 0 and 37.5 mL/kg at 120 min and 240 min into the infusion. Arterial blood samples were collected for baseline tracer enrichments and at the end of the 360-min infusion period. Blood was centrifuged at 2000 x g for 10 min at 4°C and the plasma was removed and frozen at -70°C. To measure crypt cell proliferation, 5-bromo-deoxyuridine (BrdU) was injected (50 mg/kg body) via the arterial catheter at 120 min before killing the pigs. Pigs were killed at the end of the infusion with an overdose of sodium pentobarbital (200 mg/kg), and tissues were collected as described previously (16) . Briefly, the small intestine was removed, flushed with ice-cold saline and divided into two equal segments; the proximal and distal halves were designated as jejunum and ileum, respectively. Sections from the proximal region of each segment were fixed in a buffered formalin solution for histology, and additional tissue was frozen in liquid nitrogen and kept at -70°C.

Analyses of plasma and tissue.

The IGF-I concentration in plasma samples was determined using a two-site immunoradiometric assay (IRMA; Diagnostic Systems, Webster, TX). The IGF-I IRMA assay involves an acid-ethanol extraction of the sample and recognizes both the human and porcine IGF-I peptides, which have an identical amino acid sequence. Aliquots of the control formula and the IGF-I-supplemented formulas were centrifuged at 20,000 x g for 15 min at 4°C, and the IGF-I concentration in the supernatant was measured using the IGF-I IRMA. All samples of plasma and formula were analyzed in one assay with a CV of 6.5%. The immunoisolation, purification and quantification of LPH polypeptides have been described previously (9 ,27) . BB membranes were prepared by magnesium chloride precipitation as described previously (28) . Lactase activity in the intestinal tissue homogenate and BB membranes was measured as described previously (27) . All intestinal tissue homogenization buffers contained protease inhibitors (348 g/L PMSF, 25 mg/L leupeptin, 25 mg/L aprotinin; Sigma Aldrich Chemical, St. Louis, MO). The intestinal tissue protein and DNA assays, villus morphometry measurement and immunohistochemical assay of BrdU-positive crypt cells have also been described previously (29) . Crypt cell proliferation was expressed as the number of BrdU-positive stained enterocytes expressed as a percentage of the total cells in the crypt.

The preparation of plasma, mucosal-free and mucosal protein-bound amino acid fractions and LPH polypeptides for GC-MS analysis has been described previously (15 ,27) . Mass spectrometry was conducted with the n-propyl ester heptafluorobutyramide derivative using methane negative chemical ionization as previously described (15) . The analyses were performed with a 5890 series II gas chromatograph linked to a model 5989B (Hewlett-Packard, Palo Alto, CA) quadrupole mass spectrometer. The isotope ratios were calculated using the abundance of ions at a mass-to-charge ratio of 349, 350 and 352 for leucine isotopomers and 383, 384, 388, 389 and 392 for the isotopomers of phenylalanine. The data on the ion abundances of each amino acid isotopomer were converted to tracer-to-tracee ratio expressed as mole percent (mol%) by the matrix method.

Calculations.

The details of the isotopic approach and calculations are discussed extensively by Dudley et al. (15) . Briefly, the principle of the method is that by substituting the relative isotopic enrichments of multiple tracer forms of the same amino acid that have been infused for different periods of time, the kinetics of equilibration and incorporation into both the free and protein-bound amino acid pools can be discerned. In other words, the method substitutes multiple tracers for multiple time points. In the present study, we used a simplified approach to the calculation, the modeling being carried out with the numerical routine of SAAM II (SAAM Institute, University of Washington, Seattle, WA). The fit involved weighting, using the pooled standard deviation of the respective isotopic enrichments at each time point.

In the initial analysis, the tracer enrichments of the three phenylalanine and two leucine tracers were normalized to a standard infusion rate of 20 µmol · kg^-1 · h^-1 and combined by multiplying the tracer enrichments of the phenylalanine isotopomers by the ratio of the measured plasma phenylalanine:leucine flux, which was 0.37. Values of the isotopic enrichments of the plasma and mucosal free amino acid pools in individual animals were then fitted to the equation:

(1)

in which P is the predicted isotopic enrichment at steady state, t the time of isotope administration (min) and k the rate constant of equilibration. The values for the plateau labeling and the rate constants shown in the tables are means ± SD calculated from the data for each animal, rather than those calculated from pooled labeling data. The kinetics of pro-LPH synthesis then were calculated by fitting the ratio of the isotopic enrichments of pro-LPH: free amino acid to the equation:

(2)

in which y is the ratio of the isotopic enrichments of the precursor and product, t is time (min), R the predicted steady-state ratio and b a time delay (min). Because there was a long (>100-min) delay in the appearance of tracer in BB-LPH, the precursor isotopic enrichment used in the calculation of the BB-LPH fractional synthesis rate was that of pro-LPH predicted by ^{Eq. 2} at time t—the predicted delay in the appearance of tracer in BB-LPH. Once again, the data from each animal were analyzed separately. Thereafter, the fractional synthesis rate of BB-LPH was calculated from the slope of the line of the ratio of the isotopic enrichment of BB-LPH to that of pro-LPH.

The absolute rates of synthesis of pro- and BB-LPH were calculated from the product of their respective pool sizes and their respective rate constants of synthesis. The pool size of each protein was calculated as the product of the lactase activity and the fractional contribution of pro-LPH and BB-LPH to the total lactase activity (10) . This is because purified standards for pro-LPH and BB-LPH are not available and, thus, direct measurements of their protein content from the SDS-PAGE are not possible. In effect, the results of mass and synthesis are expressed in arbitrary units and represent the amount of the isoform per gram of tissue protein and rate of synthesis per day. Thus, the ratio of the absolute rates of synthesis of BB-LPH to that of pro-LPH measures the efficiency with which newly synthesized pro-LPH is converted to the BB form of the enzyme.

Statistical analysis.

Data were analyzed by two-way ANOVA with treatment (control, low IGF, high IGF) and site of intestine (jejunum vs. ileum) as main effects. Results are expressed as means ± pooled SD unless otherwise indicated. Differences with probability values of <0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The daily formula intakes (338 ± 7 mL/kg, 12.4 ± 0.2 g protein/kg and 874 ± 16 kJ/kg) were not different among the three treatment groups. The initial body weights (1.64 ± 0.15 kg), final body weights (4.26 ± 0.33 kg) and daily weight gains (188 g/d) also were not different among the three treatment groups.

Formula and plasma IGF-I concentrations.

The cow’s milk formula contained a small amount of immunoreactive IGF-I (3.38 ± 0.22 nmol/L) compared with that in the low IGF (113 ± 8 nmol/L) and high IGF (1795 ± 84 nmol/L) formulas. This resulted in significantly (P < 0.01) different calculated mean daily IGF-I intakes (nmol/kg) in the control (1.13 ± 0.01), low IGF (38.0 ± 0.4) and high IGF (614 ± 18) groups. Despite the marked differences in IGF-I intake, the plasma IGF-I concentrations (nmol/L) measured after 14 d did not differ among the control (18.7 ± 4.1), low IGF (16.4 ± 6.0) and high IGF (14.6 ± 2.9) groups.

Intestinal mass and morphometry.

The masses of wet tissue, protein and DNA were significantly higher in the ileum than in the jejunum (Table 1 ). In addition, when pooled across treatments, the ratio of protein to DNA was 40% greater (P < 0.01) in the ileum (30.5 ± 6.0) than in the jejunum (21.9 ± 2.4). The villus height and cell proliferation index were higher, whereas the crypt depth was lower in the ileum than in the jejunum (Table 2 ). There were no significant differences among the three treatment groups in the masses of the small intestinal wet tissue, protein or DNA (Table 1) or in intestinal morphometric or cell proliferation indices of mucosal growth (Table 2) .

Administration of IGF-I did not affect any of the indices of LPH activity (Table 3). However, when averaged across IGF-I treatments, the total lactase activity per segment (698 ± 262 vs. 308 ± 122) and specific enzyme activity (215 ± 71 vs. 145 ± 45) were significantly (P < 0.005) higher in the ileum than in the jejunum. However, when the measurements were confined to the LPH in isolated BB membranes, activity was lower in the ileum than in the jejunum (Table 3) .

There were no differences among the treatment groups in either the plateau enrichments or the amino acid turnover rates in the plasma (Fig. 1 ). As expected, there was substantial dilution of the tracer amino acid in the mucosal free amino acid pool compared with plasma (Fig. 2 ). There was no segmental difference in the rate of equilibration, yet the steady-state dilution of the tracer was significantly greater in the ileum than in the jejunum. In both segments, the plateau amino acid isotopic enrichment and the ratio of the tissue to plasma amino acid labeling at plateau were significantly higher in the high IGF group than in the controls and showed a quasilinear dose relationship (Table 4).

View larger version (19K): [in this window] [in a new window]   Figure 1. Plasma tracer amino acid enrichments (mol%) in neonatal pigs fed control (n = 5), low IGF (n = 6) or high IGF (n = 6) formula. Values for each of the six different phenylalanine and leucine tracers are indicated. Values are means ± SEM.

View larger version (18K): [in this window] [in a new window]   Figure 2. Jejunum (top) and ileum (bottom) mucosal tissue free amino acid enrichments (mol%) in neonatal pigs fed control (n = 5), low IGF (n = 6) or high IGF (n = 6) formula. Values for each of the six different phenylalanine and leucine tracers are indicated. Values are means ± SEM. In jejunum, the effect of IGF-I was significant for control versus high IGF-I, P < 0.05.

In all three treatment groups, the relative enrichments of pro-LPH came to plateau values rapidly between 90 and 180 min of tracer infusion (data not shown). In the controls, the isotopic enrichment at steady state in pro-LPH was significantly higher than that of the free amino acid (i.e., the ratio of tissue to plasma was >1.0; Table 5 ). Treatment with IGF-I, mainly high IGF, was associated with a fall in the ratio of the plateau isotopic enrichments of the pro-LPH-bound and tissue-free amino acids. In both segments, the fractional synthesis rate of pro-LPH was lower in both the low and high IGF-treated groups than in the control group.

There were no significant effects of either IGF-I treatment or site of intestine on the fractional synthesis rate of total mucosal protein (Table 6). The fractional synthesis rate of BB-LPH was significantly (P < 0.01) higher in the jejunum (91 to 97% per d) than in the ileum (22–51%/d). Although IGF-I administration did not alter the fractional synthesis rate of BB-LPH in the jejunum, both low and high IGF-I treatments were associated with a more than 100% increase in BB-LPH synthesis in the ileum.

Based on scanning densitometric analysis, the relative abundances of pro-LPH in both segments were lower, and those of BB-LPH were higher in IGF-treated pigs than in controls (data not shown). The absolute synthesis rates of pro-LPH did not differ between segments, but were significantly lower in both the low and high IGF-treated pigs than in controls (Table 7). In control pigs, the absolute synthesis rate of BB-LPH in the ileum was significantly lower than in the jejunum and as a result, the efficiency of processing of newly synthesized pro-LPH to the BB was lower in the more distal region of the small intestine. The high IGF-I treatment increased the processing efficiency from 14% to 40% in the jejunum and from 4% to 19% in the ileum.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In contrast to previous findings of Houle et al. and others (19 ,26) , we found no significant effect of oral IGF-I on LPH activity, whether expressed as total activity per region, as specific activity per unit of mucosal protein or as per unit of BB protein. In addition, oral IGF-I had no effect on any measures of intestinal tissue mass, crypt cell proliferation or villus height, a finding that contrasts with some studies in neonatal pigs (16 ,19 ,26) but confirms other reports in both neonatal pigs and rodents (23 24 25) .

The explanation for the discrepancy between our findings and those of Houle et al. (19 ,26) is not clear. The low dose of IGF-I in the present study was similar to the high dose used by Houle et al. (19) , and the ages and period of treatment were similar. However, there were some potentially important differences in the respective experimental protocols. First, we used vaginally born pigs that had suckled on the dams for 24–36 h before entering the study whereas Houle et al. (19 ,26) used cesarean-derived, colostrum-deprived pigs. Another, perhaps critical difference between the two studies was that in the present study the intestinal tissue used to assess lactase expression and synthesis was obtained in fed pigs, whereas Houle et al. (19 ,26) studied pigs after overnight food deprivation. In the present study, we reasoned that is was most logical to test the effect of oral IGF-I on lactase processing in the fed rather than the overnight food-deprived state, because that is when both the growth factor and the substrate (i.e., lactose) are presented to the mucosal epithelium. Given the present findings, if indeed lactase activity is only up-regulated by oral IGF-I in the food-deprived state, then the response to oral IGF-I is of limited relevance to the functional capacity to digest lactose.

Despite the lack of an effect on intestinal protein mass or LPH activity, oral IGF-I had a number of effects on LPH synthesis and processing. First, chronic administration of oral IGF-I suppressed the fractional and absolute synthesis rates of pro-LPH and lowered the relative contribution of the pro-peptide to total immunoprecipitable LPH. At the same time, IGF-I treatment increased the rate of mature BB-LPH synthesis, particularly in the ileum. Because the rate of pro-LPH synthesis was not increased by IGF-I, the increase in BB-LPH synthesis reflected a substantial increase in the proportion of newly synthesized pro-LPH that was processed and inserted in the BB membrane. The net effect of these two opposing kinetic responses was no change in the calculated mass of BB-LPH, a conclusion that is consistent with the LPH-specific activity measurements. The mechanism for decreased pro-LPH synthesis is unknown and contrary to the previous finding that oral IGF-I increased mRNA abundance (26) . In the jejunum, there was a IGF-I dose-dependent increase in the mucosal free pool tracer enrichment. In addition, the mucosal free pool tracer enrichment was much higher in the jejunum than in the ileum. Given that there were no differences in the circulating arterial plasma tracer enrichment, there are three possible explanations for these differences in the mucosal free pool tracer enrichment: 1) IGF-I increased the rate of basolateral transport of tracer amino acid from the arterial circulation into the cell, 2) IGF-I increased the rate of apical transport of unlabeled tracee amino acid from the intestinal lumen into the cell, or 3) IGF-I suppressed the rate of intracellular proteolysis and release of unlabeled tracee amino acid. Based on evidence that IGF-I has been shown to increase amino acid transport (31) , it is conceivable that the increased mucosal tracer enrichments resulted from increased basolateral transport from the circulation. However, if IGF-I were to increase the apical transport of amino acid from the lumen into the epithelial cells, then this would dilute or decrease the tracer enrichment. However, we found it to be increased by IGF-I, making this second possibility seem unlikely. The third, and in our opinion most likely, possibility is that IGF-I suppressed proteolysis and the intracellular release of unlabeled amino acid, which led to the increased mucosal tracer enrichments. The same holds true for the regional differences along the intestine, suggesting higher rates of proteolysis in the ileum than in the jejunum. In support of this interpretation, the processing efficiency of pro-LPH to BB-LPH was consistently and positively correlated with the mucosal free tracer enrichments with respect to both IGF-I and regional site differences. Thus, we postulate that the increased processing efficiency to BB-LPH is a likely consequence of suppressed intracellular proteolysis.

Although the focus of this study was IGF-I, perhaps the most remarkable observations were the differences in the kinetics of LPH synthesis and processing between the jejunum and ileum. The first observation of note was that the total segment and tissue-specific activity of lactase in all treatment groups was between 50% and 100% higher in the ileum than in the jejunum. The differences between segments in both of these measures of lactase activity were associated with substantially longer villi. A longer villus implies a longer enterocyte lifespan, and, hence, a greater number of differentiated enterocytes expressing lactase activity. Interestingly, however, the lactase activity measured in the BB fraction was significantly lower in the ileum than in the jejunum. Consistent with BB activity measurements, we found that the absolute and fractional synthesis rates of BB-LPH were also significantly slower in the ileum than in the jejunum. Thus, it seems that although there is a much greater mass of lactase activity in the ileum, the activity of lactase per villus enterocyte is much lower than in the jejunum, reflecting a much lower efficiency of processing of pro-LPH to BB-LPH form. Of additional interest is the fact that our estimates of the efficiency of LPH processing are consistent with the reports implicating posttranslational control in the general decline in the activity of LPH, despite elevated expression of LPH mRNA, along the proximal-to-distal axis of the intestine (11 ,12) .

In summary, the current results demonstrate that chronic oral IGF-I supplementation significantly decreases the synthesis of pro-LPH but increases the efficiency of its posttranslational processing to BB-LPH, resulting in increased BB-LPH synthesis in the ileum. Despite this, however, all measurements of intestinal lactase activity were unaffected by oral IGF-I treatment. Our current findings confirm several previous reports, which together strongly suggest that oral IGF-I supplementation does not affect any aspect of intestinal growth or morphology in healthy well-nourished neonates.

	ACKNOWLEDGMENTS

 
We thank Xiaoyan Chang and Judy Rosenberger for the technical assistance. We also thank Jane Schoppe for assistance in preparation of the manuscript and Leslie Loddeke for editorial assistance.

	FOOTNOTES

 
¹ The contents of this publication do not necessarily reflect the views or policies of the U. S. Department of Agriculture and mention of trade names, commercial products or organizations does not imply endorsement by the U. S. Government.

² Supported by the U. S. Department of Agriculture Agricultural Research Service under Cooperative Agreement 58-6250-6001.

⁴ Present address: Ming Z. Fan, Department of Animal and Poultry Science, Room 250, Animal Science/Nutrition Building, University of Guelph, Guelph, Ontario, Canada N1G 2W1.

⁵ Abbreviations used: BB-LPH, brush-border lactase-phlorizin hydrolase; IGF-I, insulin-like growth factor I; IRMA, immunoradiometric assay; LPH, lactase-phlorizin hydrolase; pro-LPH, prolactase-phlorizin hydrolase.

Manuscript received March 2, 2001. Initial review completed April 13, 2001. Revision accepted May 30, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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