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Am J Physiol Regul Integr Comp Physiol 283: R1450-R1460, 2002. First published August 29, 2002; doi:10.1152/ajpregu.00172.2002
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Vol. 283, Issue 6, R1450-R1460, December 2002

IGF alters jejunal glucose transporter expression and serum glucose levels in immature rats

Robert H. Lane1,*, Bohuslav Dvorak2,*, Nicole K. MacLennan1, Katerina Dvorakova3, Melissa D. Halpern2, Tho D. Pham1, and Anthony F. Philipps4

1 Department of Pediatrics, David Geffen School of Medicine at University of California Los Angeles, Mattel Children's Hospital, Los Angeles, California 90095-1752; 2 Department of Pediatrics, the University of Arizona College of Medicine, Steele Memorial Children's Research Center, and 3 Department of Microbiology and Immunology, the University of Arizona College of Medicine, Tucson, Arizona 85724-5073; and 4 Department of Pediatrics, University of California at Davis, Sacramento, California, 95817


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Milk-borne insulin-like growth factors (IGFs) enhance nutrient absorption in the immature intestine, which is characterized by low levels of glucose oxidation. We therefore hypothesized that feeding a rat milk substitute (RMS) devoid of growth factors to rat pups would lower serum glucose levels relative to dam-fed control rats and that supplementation of RMS with physiological doses of either IGF-I or IGF-II would normalize serum glucose levels via increased jejunal glucose transporter 2 (GLUT2) and high-affinity Na+-glucose cotransporter (SGLT1) expression. We found lower serum glucose concentrations in RMS-fed pups; in contrast, serum glucose levels in the IGF-supplemented pups were similar to those of dam-fed controls. RT-PCR and laser scanning confocal microscopy similarly demonstrated that IGF supplementation increased expression of jejunal glucose transporters. Further experiments demonstrated that IGF supplementation altered mRNA levels of key mitochondrial enzymes without altering jejunal lactase activity. We conclude that IGF-I and IGF-II supplementation increases serum glucose levels in the immature rat pup fed artificial formula and alters gene expression of the jejunal glucose transporters.

SGLT1; GLUT2; GLUT1; fatty acid oxidation; glucose oxidation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN MILK contains the peptides insulin-like growth factor-I (IGF-I) and IGF-II (13, 14). IGF-1 and IGF-II affect multiple cellular processes including glucose uptake (23, 55). Parenteral administration of IGF-I increases jejunal glucose absorption in adult rats, and in neonatal piglets, oral IGF-I increases jejunal 3-O-methylglucose uptake (2, 64).

Transepithelial uptake and transfer of glucose in the immature intestine predominantly involves two protein transporters (61). The first is the high-affinity Na-glucose cotransporter (SGLT1), which transports glucose across the enterocyte apical brush-border membrane. The second, glucose transporter 2 (GLUT2), transports glucose across the enterocyte basolateral membrane. A third transporter, GLUT1, is also found in immature rat intestine, although its function is unknown (47). Gene expression of all three glucose transporters is subject to ontogenic variation. GLUT1 expression decreases after birth, and SGLT1 and GLUT2 expression increase postnatally through the suckling period and peak after weaning (47, 61).

Transepithelial intestinal glucose transport is a key function of suckling rat intestine that provides energy substrate to multiple tissues, particularly the brain. Intestinal glucose oxidation is relatively low in the suckling rat as a result of the high intramitochondrial [NADH]-to-[NAD+] ratio that characterizes the immature intestine, which may have the effect of sparing this substrate for nongastrointestinal tissues (33-35). A high intramitochondrial [NADH]-to-[NAD+] ratio has been previously associated with increased expression and function of two key enzymes of fatty acid oxidation, carnitine palmitoyl transferase I (CPTI) and the trifunctional protein of beta -oxidation (HADH) (41). CPTI catalyzes the exchange of acylcarnitines across the mitochondrial outer membrane and is a rate-limiting step of mitochondrial beta -oxidation (48). HADH directly competes with Krebs cycle dehydrogenases such as isocitrate dehydrogenase (ICD) for NAD+, and as a result, increased expression of HADH could contribute to the low level of intestinal glucose oxidation (18). Interestingly, parenteral administration of IGF-I increases lipid oxidation as well as CPTI gene expression and activity (21, 26).

In the neonatal rat intestine, orogastrically administered IGF-I and IGF-II are stable and biologically active; moreover, in a study in which immature rats were artificially reared on a rat milk substitute (RMS) diet devoid of growth factors, those given RMS supplemented with IGF-I had improved weight gain and brain growth (52-54). We therefore hypothesized that RMS supplementation with IGF would increase serum glucose levels in conjunction with increased SGLT1 and GLUT2 intestinal expression in immature rats vs. pups fed an unsupplemented RMS. We further hypothesized that IGF supplementation would increase CPTI and HADH mRNA levels relative to ICD in supplemented vs. unsupplemented rats.

To test this hypothesis, we fed rat pups on day 8 through day 12 of life either RMS or RMS supplemented with IGF-I or -II in concentrations similar to those in native rat milk or colostrum. Blood glucose levels and midjejunal lactase levels were quantified in association with measurements of SGLT1, GLUT2, GLUT1, CPTI, HADH, ICD, and glutamate dehydrogenase (GD) mRNA levels. Histological localization of GLUT2 and SGLT1 in the midjejunum was performed using laser confocal microscopy to characterize further the effect of the enteral IGF supplementation on developing intestine. GD mRNA levels were measured because glutamine is the predominant oxidative fuel of the immature intestine, and in contrast to glucose, oxidation of this substrate is not significantly affected by fluctuations of the mitochondrial [NADH]-to-[NAD+] ratio (32).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. Recombinant human (rh) IGF-I and rhIGF-II were obtained from Kabi Pharmacia Biosciences Center (Stockholm, Sweden). The RMS was prepared as previously described by Dvorak and Stepankova (16). The IGFs were added to the RMS in the following manner. Sterile plastic scintillation vials (20-ml size, Research Products International, Mt. Prospect, IL) were used to mix and store the RMS + IGF mixtures. Reconstituted IGF (1-2 µg in 0.5 ml of 0.1 M acetic acid) was added to each vial, and the contents were lyophilized and stored at -20°C until use. Each morning during the experimental period, 0.2 ml of Tris buffer (50 mM, pH 7.4) was added to each vial and then shaken using a standard laboratory shaker (Vortex Genie, Fisher Scientific, Bohemia, NY). Freshly prepared RMS was subsequently added to each vial to achieve final IGF-I and IGF-II concentrations of 50 and 100 ng/ml, respectively.

Animals. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Arizona (A-324801-95081) and are in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society (3). Individual litters of Sprague-Dawley rats raised in our colony were used in these experiments. All litters were culled to 10 pups on the second postnatal day to ensure uniformity in growth rate, including mother-fed pups. Beginning on day 8 of life, rat pups within each litter were anesthetized with the use of halothane (Halocarbon Laboratories, River Ridge, NJ). A polyethylene catheter with a hook-shaped end (PE-20, Clay Adams/Becton Dickinson, Parsippany, NJ) was inserted into the stomach of each suckling using a 16-gauge Cathlon IV Catheter Placement Unit (Critikon/Johnson & Johnson, Tampa, FL), as previously described (44). The catheter was then affixed to each rat pup by means of subcutaneous suturing with the catheter exiting at the thoracic vertebral area. During and after surgery, pups were kept warm on heating pads. After postsurgical recovery, which included a 2-h fast, pups were placed in plastic cups containing Bed O'Cobs corncob bedding (The Anderson's Management, Maumee, OH) and floated in a 39°C water bath (Precision, Chicago, IL) for the duration of the feeding study to maintain the ambient temperature and humidity. The gastrostomy tubes were connected to syringes on model 44 Harvard infusion pumps (Harvard Apparatus, South Natick, MA) in a refrigerator by means of Silastic tubing (Dow Corning, Midland, MI). After gastrostomy, neonatal rats were weighed and the volume of diet to be given to each animal was calculated to deliver ~37% of body weight per 24-h period (17). Sucklings were randomized to each of the four diets, and average pup weights in these groups were equal.

Suckling rats then received a 2.5% glucose plus electrolyte solution (Pedialyte, Ross Laboratories, Columbus, OH) for 4-6 h to assess postoperative viability and catheter patency. Syringes containing Pedialyte were then removed and replaced with those containing RMS, RMS + IGF-I, or RMS + IGF-II diets. Catheter distance from refrigerated syringe to gastrostomy allowed for gradual formula warming to room temperature. Rat pups were examined each morning, and the perianal region was stroked to promote micturition and defecation. Daily weight was also recorded for each day.

After 96-h infusion of RMS with or without IGF-I or IGF-II, respectively, the rat pups were weighed and the tail length from tip to anus was measured for each. The pups were then killed by decapitation, and the trunk blood was removed. Blood was allowed to clot at room temperature, and serum was removed and stored at -70°C for later assay. The small intestine was isolated from each animal (from duodenum to cecum) and weighed, and its length was measured. The middle third from each intestine (midjejunum) was removed and treated as noted below. Mother-fed pups were also killed, and samples were taken in a similar manner at 12 days of age after suckling to mimic the "fed" state of the artificially reared pups.

Serum glucose levels. Serum glucose concentrations were measured using a glucose analyzer and the glucose oxidase method (Beckman Instruments, Palo Alto, CA).

Midjejunal protein and DNA content. Midjejunal homogenates were prepared using a 1:4 (wt/vol) ratio of tissue in 50 mM Tris (pH 7.0) and a Polytron tissue homogenizer (Brinkman Instruments, Westburg, NY). Each homogenate was centrifuged at 4,000 g for 10 min at 4°C, and the supernatant was then stored for later assay. Protein and DNA contents of samples were assayed using standard methods (46); n = 6 for dam-fed, RMS + IGF-I, and RMS + IGF-II study groups from separate litters, and n = 4 for the RMS group.

Midjejunal specific lactase levels. Midjejunal lactase specific activities were assayed by the method of Asp et al. (4). A total of eight animals for the dam-fed and RMS + IGF-II groups, and six animals from the RMS and RMS + IGF-I groups, respectively, were used and represented three litters additional to those utilized for the other portions of the study. Briefly, aliquots of midjejunal homogenate were incubated at 37°C for 1 h in the presence of a substrate consisting of lactose with parahydroxymercuribenzoic acid to inhibit acid galactidase activity, and the glucose production was measured with glucose oxidase. The specific activity of lactase is expressed as micromoles of substrate hydrolyzed per milligram protein per hour.

RNA isolation. Total RNA was isolated from tissue using the RNeasy Mini Kit (Qiagen, Santa Clarita, CA) as previously described (17). All RNA samples were incubated with RNase-free DNase enzyme (20 U per reaction) for 10 min at 37°C to eliminate DNA contamination. The RNA concentration was quantified by UV spectrophotometry at 260 nm (A260), and the purity was determined by the A260/A280 ratio (SPECTRAmax PLUS, Molecular Devices, Sunnyvale, CA). The integrity of RNA samples was verified by electrophoresis on 1.2% agarose gel containing formaldehyde (2.2 M) and ethidium bromide in 1 × MOPS buffer [40 mM MOPS (pH 7.0), 10 mM sodium acetate, and 1 mM EDTA (pH 8.0)].

RT-PCR. This methodology of RT-PCR has been previously reported (38-43). cDNA was synthesized using random hexamers and SUPERSCRIPT II RT (Life Technologies, Gaithersburg, MD) from 1.0 µg of midjejunal RNA added to 0.01 µg of bovine retinal RNA. The resulting cDNA was resuspended in 20 µl of water, diluted into 1:100 aliquots, and stored at -20°C until use. Jejunal mRNA extracts from five animals per experimental group were used from five separate litters. Amplification primers for SGLT1, GLUT2, GLUT1, CPTI (liver isoform), HADH (alpha -subunit), ICD, GD, and rhodopsin are listed in Table 1. Each reaction was carried out in a total volume of 20 µl using 5 µl of diluted RT reaction product, 20-30 pM of rhodopsin primers, 10-50 pM of target primers, 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 1.5 mM MgCl2, 20 µmol dNTP, 0.2 µCi of [alpha -32P]dCTP (3,000 mCi/ml: 1.0 mCi = 37 MBq), and 0.1 U of AmpliTaq Gold polymerase (Perkin-Elmer, Norwalk, CT). Amplification of the rat target cDNA required a different ratio of rhodopsin DNA primers to target DNA primers because of variation in target DNA secondary structure as well as differences in relative abundance. To determine reaction conditions when both amplicons were simultaneously produced exponentially, we reverse transcribed and amplified serial dilutions of rat RNA with standard amounts of retinal RNA under different conditions and cycle numbers. Once optimal conditions were determined, we ran a single standard serial dilution with each quantification to verify parallel production of both rat and bovine PCR products. This standard serial dilution consisted of cDNA reverse transcribed from 0.5, 1.0, and 2.0 µg of rat skeletal muscle control RNA, respectively, and 0.01 µg of bovine retinal RNA. Reactions were replicated three times once optimal PCR conditions were determined, and the primer concentrations were identical between study groups for each rat DNA target, respectively.

                              
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  		Table 1.   Sequences of each primer pair and their location in sequences cited in GenBank

PCR amplification was carried out under the following conditions: one initial denaturation cycle of 94°C for 9 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. A final extension at 72°C for 10 min completed the amplification. The PCR products were separated on a nondenaturing 5% acrylamide gel, and the radioactivity incorporated into the amplified product was quantified using a phosphorimager and Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). The relative abundance of SGLT1, GLUT2, GLUT1, CPTI, HADH, ICD, and GD was quantified relative to that of a control rhodopsin band from the same reaction, which was assigned an arbitrary level of unity.

Immunohistology of the SGLT1 and GLUT2. Samples of midjejunum were fixed overnight in 70% ethanol, processed, paraffin-embedded, and microtome-sectioned at 4-6 µm. Sections were deparaffinized in xylene and rehydrated in a graded series of ethanol dilutions. Sections were blocked with 5.0% BSA (Vector Laboratories, Burlingame, CA) for 10 min and then incubated with either 1 µg/ml rabbit anti-GLUT2 polyclonal antibody (Biogenesis, Kingston, NH) or 1 µg/ml anti-SGLT1 polyclonal antibody (Alpha Diagnostic International, San Antonio, TX) for 60 min. After three PBS washes, biotinylated rabbit anti-goat secondary antibody (Vector Laboratories) was applied for 60 min, followed by incubation with Cy5-conjugated streptavidin for 1 h. Nuclei were labeled after RNase digestion using YoYo-1 (Molecular Probes, Eugene, OR), as previously described (11). Coverslips were then mounted using mounting media (DAKO, Carpinteria, CA), and slides were stored at 4°C. A laser scanning confocal microscope (LEICA TSD-4D, Heidelberg, Germany) equipped with an argon-krypton laser was used to obtain images. The laser power, the voltage of the photomultiplier tube, and the number of line scans were constant so that fluorescent intensities of various samples could be compared. Control sections were treated with the same procedures except they were incubated with 1% BSA instead of the specific antibodies. No signal was observed in controls. Sections from dam-fed, RMS, RMS + IGF-I, and RMS + IGF-II groups were simultaneously stained so that comparisons of staining intensities between groups could be assessed.

Statistics. All data are represented as means ± SE. Statistical differences between serum blood glucose levels, animal and tissue morphological features, midjejunal protein/DNA content, and lactase specific activities were assessed using one-way ANOVA and Fisher's protected least significant difference. For RT-PCR, statistical analyses were performed using the nonparametric Wilcoxon matched-pair test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Somatic and intestinal growth. As noted in Table 2, body weight and tail lengths between treatment groups were all statistically similar to those of the dam-fed controls. However, small intestinal weights and lengths in animals fed RMS were significantly greater than those of matched dam-fed controls. Addition of IGF-I and IGF-II was not associated with further increments in intestinal weight or length.

                              
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  		Table 2.   Body and intestinal growth in suckling rats

Serum glucose levels. Serum glucose concentrations in RMS-fed rat pups (105 ± 13 mg/dl, n = 5) were significantly (P < 0.05) below those of dam-fed controls (180 ± 21 mg/dl, n = 6). Supplementation of RMS with physiological amounts of IGF-I or -II resulted in serum glucose levels (153 ± 10, n = 6, and 150 ± 4 mg/dl, n = 6, respectively) significantly above those of the RMS-fed animals and statistically similar to those of the dam-fed pups (Fig. 1).


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  		Fig. 1.   A: quantification of blood glucose level, midjejunal activity, high-affinity Na+-glucose cotransporter (SGLT1) mRNA, and glucose transporter 2 (GLUT2) mRNA in 12-day-old pups fed rat milk substitute (RMS), RMS + insulin-like growth factor (IGF)-I, or RMS + IGF-II. Data are expressed as mean percentage ± SE relative to levels found in dam-fed (DF) pups (* P < 0.05 vs. DF pups; # P < 0.05 vs. RMS-only pups). B: representative phosphorimages of SGLT1 and GLUT2 RT-PCR products from midjejunum of DF pups or pups fed either rat RMS, RMS + IGF-I, or RMS + IGF-II. The top band is the rat target (SGLT1 or GLUT2), and the bottom band is the rhodopsin control. RT-PCR products were quantified by phosphorimage analysis (Molecular Analyst software, Bio-Rad).

Midjejunal protein and DNA content. Midjejunal protein content did not differ between the 12-day-old pups fed RMS vs. the pups in either supplemented RMS group (RMS, 71.6 ± 8 mg/g tissue; RMS + IGF-I, 85.8 ± 8.5 mg/g tissue; RMS + IGF-II, 78.7 ± 10.5 mg/g tissue). Midjejunal protein content of all pups fed RMS was less than those pups that were mother fed (dam fed = 97.13 ± 9.3 mg/g tissue).

Similarly, midjejunal DNA content did not differ between the 12-day-old pups fed RMS vs. either supplemented RMS group (RMS, 3.54 ± 0.4 mg/g tissue; RMS + IGF-I, 4.39 ± 0.8 mg/g tissue; RMS + IGF-II, 3.66 ± 0.4 mg/g tissue). DNA contents for both RMS and RMS + IGF-II groups were significantly less than pups that were mother fed (dam fed, 5.90 ± 0.6 mg/g tissue).

Lactase specific activities. Midjejunal lactase activities were depressed (3.00 ± 0.23 µmol · mg protein-1 · h-1) in RMS-fed animals compared with dam-fed controls (5.48 ± 0.40 µmol substrate · mg protein-1 · h-1, P < 0.05). Although IGF supplementation was associated with a modest increase in lactase activity (3.47 ± 0.27 and 3.62 ± 0.52 µmol substrate · mg protein-1 · h-1 for IGF-I- and IGF-II-supplemented rats, respectively), these changes were not statistically different from RMS-fed animals.

Glucose transporter mRNA levels. Midjejunal SGLT mRNA levels were significantly lower in RMS-fed rat pups compared with dam-fed controls (Fig. 1). Midjejunal SGLT mRNA levels were significantly increased in rat pups fed either RMS + IGF-I or RMS + IGF-II vs. those pups fed only RMS. The differences in SGLT mRNA levels between RMS + IGF-I, RMS + IGF-II, and dam-fed groups were not significant. Similarly, GLUT2 midjejunal mRNA levels were increased in the rat pups fed the supplemented RMS, and these levels approached those in the dam-fed pups (Fig. 1).

Midjejunal GLUT1 mRNA levels were similar in dam-fed pups and those fed RMS + IGF-II formula. There were no significant differences in GLUT1 mRNA levels between pups fed RMS and RMS + IGF-I [RMS, 100 ± 8%; RMS + IGF-I, 136 ± 16%; RMS + IGF-II, 233 ± 23% (P < 0.05 vs. RMS group); dam fed, 303 ± 40% (P < 0.05 vs. RMS group)].

Histological localization of GLUT2 and SGLT1. Because traditional immunohistological techniques resulted in diffuse signals for the presence of GLUT2 and SGLT1, more sensitive laser scanning confocal microscopy was employed to localize these proteins. Histological examination of midjejunum samples from dam-fed pups showed very strong GLUT2 signals localized throughout the epithelial cells of villi, with the largest concentration occurring in the basolateral area of the epithelial cells (Fig. 2A). In contrast, GLUT2 expression in the RMS group was significantly decreased with low to no staining in midjejunal epithelium (Fig. 2B). The pattern of GLUT2 localization and the signal density in the RMS + IGF-I and RMS + IGF-II groups (Fig. 2, C and D, respectively) were similar to that observed in the dam-fed group.


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  		Fig. 2.   GLUT2 histological localization in midjejunum of 12-day-old rats. Laser scanning confocal localization microscopy of representative sections from DF, RMS, RMS + IGF-I, and RMS + IGF-II groups are shown. A: DF group, with strong GLUT2 signal localized predominantly in the basolateral membrane and cytoplasm of the villous epithelial cells. B: RMS group, with markedly decreased signal with low to no staining. RMS + IGF-I (C) and RMS + IGF-II groups (D) display strong signal similar to DF group. E: control section treated with the same procedure except for incubation with 1% BSA instead of GLUT2 primary antibody. Magnification, ×200.

Histological localization of SLGT1 in the dam-fed group revealed the pattern of scattered cells with positive signal (Fig. 3A). In the RMS group, no SGLT1-positive cells were observed (Fig. 3B). In contrast, animals fed with either RMS + IGF-I or RMS + IGF-II exhibited similar patterns of SGLT1 signal as detected in the dam-fed group (Fig. 3, C and D, respectively).


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  		Fig. 3.   SGLT1 histological localization in midjejunum of 12-day-old rats. Lasers scanning confocal microscopy of representative sections from DF, RMS, RMS + IGF-I, and RMS + IGF-II groups are shown. A: DF group with isolated SGLT1-positive signal in scattered cells (arrows). B: RMS group with minimal to no SGLT1-positive signal observed. RMS + IGF-I (C) and RMS + IGF-II groups (D) display SGLT1-positive signal in scattered cells (arrows) comparable to DF group. E: control section treated with the same procedure except for incubation with 1% BSA instead of GLUT2 primary antibody. Magnification, ×200.

Mitochondrial dehydrogenase and CPTI mRNA levels. Midjejunal mRNA levels of HADH and CPTI were similar between dam-fed rat pups and pups fed either RMS + IGF-I or RMS + IGF-II vs. those pups fed RMS only (Fig. 4). In contrast, midjejunal mRNA levels of ICD were significantly decreased in all three groups compared with the RMS-only group (Fig. 4). GD mRNA levels were not significantly altered in any of the groups (Fig. 4).


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  		Fig. 4.   A: quantification of midjejunal carnitine palmitoyl transferase I (CPTI), trifunctional protein of beta -oxidation (HADH), isocitrate dehydrogenase (ICD), and glutamate dehydrogenase (GD) mRNA levels in 21-day-old pups fed RMS, RMS + IGF-I, or RMS + IGF-II. Data are expressed as mean percentage ± SE relative to levels found in DF pups (* P < 0.05 vs. DF pups; # P < 0.05 vs. RMS-only pups). B: representative phosphorimages of CPTI, HADH, ICD, and GD RT-PCR products from midjejunum of DF pups or pups fed RMS, RMS + IGF-I, or RMS + IGF-II. The top band is the rat target (CPTI, HADH, ICD), and the bottom band is the rhodopsin control with the exception of GD. RT-PCR products were quantified by phosphorimage analysis (Molecular Analyst software, Bio-Rad).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IGFs are potent mitogenic growth factors thought to play a significant role in growth regulation in the perinatal period and beyond (55). Whether or not such growth factors, when ingested in milk, have significant effects on the suckling's development is unclear but has been suggested by a variety of studies (1, 2, 14, 17, 52). In the present work, the use of an artificial formula devoid of peptide growth factors was associated with a diminution in serum glucose concentration in suckling rats.

An initial study using RMS by Auestad et al. (5) found a 20% decrease in blood glucose levels, although this study was limited by a small number of animals and the findings were not statistically significant (n = 4). The degree of hypoglycemia the animals in the study of Auestad et al. (5) experienced may also have been limited by the age of the animals that were studied, which was up to 17 days of life, because the ability to maintain glucose homeostasis matures throughout the perinatal and suckling periods (19, 36). Similarly, a study by Kinouchi et al. (37) did not find evidence of hypoglycemia in animals that were studied up to day 21 of life; moreover, this contrasting study used a different RMS that was not a cow milk-based formula (28, 37).

Enteral supplementation of RMS with physiological amounts of IGF-I or IGF-II normalized systemic blood glucose levels in association with increased mRNA levels of the intestinal transepithelial glucose transporters and altered mRNA levels of key mitochondrial enzymes. These changes occurred despite the fact that the RMS contained a similar carbohydrate (lactose) concentration as native rat milk (5). In addition, the changes observed in intestinal lactase concentration would not be expected to account for the differences observed in serum glucose levels between the RMS-supplemented and unsupplemented groups. Our data suggest that milk-borne IGFs also contribute to neonatal glucose homeostasis, which is particularly intriguing in light of the recent evidence that adult small intestine is insulin sensitive and potentially contributes to systemic glucose regulation (10). Our finding of increased intestinal gene expression is novel and suggests a mechanism by which the IGFs increase blood glucose level because intestinal mRNA levels of both transporters have been previously demonstrated to correlate with transepithelial glucose transport (56, 59, 60).

Alexander and Carey (1, 2) similarly found increased jejunal glucose uptake in neonatal piglet intestine in response to enteral IGF-I administration without a concomitant increase in lactase activity. In the latter study, the presence of IGF-1 increased in vitro jejunal glucose uptake through the activation of phosphatidylinositol-3 kinase; protein levels of jejunal SGLT1 were unchanged, however. Possible reasons for the discrepant findings include intrinsic species differences and the ages of the animals studied. In the piglet study, the period of the investigation was the first 5 days of life; in the present study, the period was day 8 to day 12 of life. Gene expression of both SGLT1 and GLUT2 increases during the suckling period (47, 61). As a result, the extent to which the jejunum may be capable of increasing SGLT1 protein levels may not be detectable by Western blotting.

Differences in the formulas between the two investigations could also contribute to the difference in SGLT1 expression. The present study used an RMS pioneered by Edmond and colleagues (5) that is characterized by a relatively high fat content (similar to natural rat milk). The study by Alexander and Carey (1) used a porcine milk replacer (Milk Specialities, Dundee, IL) that is characterized by high protein content relative to the rat formula as well as the inclusion of oxytetracycline and neomycin. Oxytetracycline inhibits mitochondrial protein synthesis, and neomycin, as well as other aminoglycoside antibiotics, potentially affects eukaryotic protein synthesis and processing (8, 12, 20, 44). The increase in glucose uptake observed by Alexander may be a result of IGF-I promoting SGLT1 translocation, a mechanism that may be contributing to the association between glucose levels and glucose transporter expression in this study. Because the Alexander study did not include immunohistochemistry, SGLT1 histological localization cannot be compared between the two studies.

The regulation of GLUT2 expression in the immature intestine is not well defined, particularly within the context of milk-borne growth factors. This is likely to be a fruitful area of future study based on the recent work of Kellett and Helliwell (30). In this study, the authors found that GLUT2 transport of glucose was up to threefold greater than that of SGLT1 and concluded that the principal route for intestinal glucose absorption is GLUT2-mediated facilitated diffusion across the brush-border membrane. Even less is known about the regulation and significance of GLUT1 in the neonatal intestine. No GLUT1 is expressed in the adult rat intestine (47). Boyer et al. (7) found that streptozotocin-induced diabetes increased enterocyte GLUT1 expression, and it may be that the observed increase in GLUT1 mRNA level correlates with serum glucose levels.

The SGLT1 and GLUT2 immunohistochemistry findings using laser confocal microscopy support our findings of increased mRNA levels of these genes and are novel secondary to the early postnatal age of the animals. The differences in histological localization between the IGF-I- and IGF-II-supplemented groups may occur because initially IGF-1 signals through tyrosine kinase-linked phosphorylation, and IGF-II signals through inhibitory G protein(s) (22, 49) .

The paucity of SGLT1 is consistent with previous reports of low levels of expression in the immature animal and suggests that either GLUT2 or paracellular transport significantly contributes to intestinal glucose absorption in the immature intestine independent of SGLT1 (29, 31). Khan et al. (31) found the glucose absorption rate in 16-day-old suckling rats to be ~10% of the glucose absorption rate of weaned rat pups. When Khan et al. (31) further treated the pups with the SGLT1 inhibitor phloridzin, glucose absorption continued although sodium absorption did not; moreover, glucose uptake was two to three times greater in the suckling pups vs. the weaned pups after the phloridzin treatment. This suggests, similar to our results, that SGLT1-independent glucose absorption may be relatively important in the immature animal.

In mature animals, SGLT1 is primarily located in the brush border. Because SGLT1 brush border localization is determined by rates of exocytosis and endocytosis, our finding of diffuse SGLT1 cellular localization may be a result of the age of the animals and secondary to an immature translocation mechanism (62). This would be similar to glucose transporter 4, whose translocation in skeletal muscle is developmentally regulated and less responsive in the immature rat.

It is noteworthy that the relative normalization in blood glucose levels occurs in the absence of an increase in midjejunal lactase specific activity. Our findings contrast those of Donovan and colleagues (24, 25), who found that IGF-I supplementation of the neonatal piglet caused a significant increase in jejunal lactase activity. A possible explanation for these conflicting results includes the dosing of IGF-I. In the piglet studies, 0.08-0.33 µg · g-1 · day-1 of enteral IGF-I was given, whereas the rat pups in the present study received ~0.012 µg · g-1 · day-1 of enteral IGF-I. The piglet studies did note a direct correlation between the amount of IGF-I given and the lactase activity (24).

Interestingly, Burrin et al. (9) noted that oral IGF-I supplementation in piglets altered posttranslational processing of lactase, but not lactase activity. Lactase activity increases in piglets that are receiving 100% total parenteral nutrition (51). IGF-I supplementation of 80% total parenteral nutrition and 20% enteral nutrition further increases lactase activity (51). Those studies that find increased lactase activity may be measuring it during a relative postabsorptive state vs. a fed state. Unfortunately, specific information regarding the relation between tissue collections and feeding states is not included in most studies.

Other differences between the two groups of studies that may explain the contrasting lactase activity levels include the duration of IGF-I supplementation (piglets, 14 days; rats, 3 days) and intrinsic species differences. In a study utilizing rats, Steeb et al. (58) also found that orogastrically delivered IGF-I did not stimulate intestinal lactase activity in neonatal rat pups.

It is also noteworthy that serum glucose levels fell in the RMS-fed vs. the dam-fed pups despite the finding that intestinal length and weight increased. Overgrowth of preweaning small intestine is an almost universal finding in artificially reared animals (15, 28, 50). These studies have speculated that the stimulus is unidentified cow's milk proteins or growth factors; however, in this study, RMS supplementation with neither IGF-I nor IGF-II exacerbated this growth. Moreover, the cow's milk-based formula with its complement of suspect proteins did not stimulate changes in glucose transporter expression, which suggests that the presence or absence of IGF is responsible for the changes in SGLT1 and GLUT2 expression

IGF-I and IGF-II are present in milk from a number of mammalian species, including humans and rats (13, 14). The developing intestine contains relatively high levels of both IGF-I and IGF-II receptors (57, 63). The type I receptor distributes predominantly in the submucosa and lamina propria, and the type II receptor locates predominately in the villous epithelium. We have previously demonstrated that enterally administered IGF-I and IGF-II are stable in the neonatal intestine and bind to their appropriate receptors (53, 54). The binding of these receptors is necessary for the initiation of the metabolic effects associated with the IGFs, and among those metabolic effects is the stimulation of lipid oxidation (6, 27, 45).

A key step in the mitochondrial lipid beta -oxidation pathway is the reduction of [NAD+], and the immature rat intestine is characterized by a high [NADH]-to-[NAD+] ratio. Previous studies in skeletal muscle demonstrate that increased mRNA levels and function of CPTI and HADH are associated with a high intramitochondrial [NADH]-to-[NAD+] ratio (33, 41). It is therefore intriguing that addition of the IGFs to RMS affects mRNA levels of these enzymes. Because the in vitro addition of palmitate inhibits glucose oxidation in preweaned rat intestine, we speculate that increased expression of CPTI and HADH in response to IGF supplementation increases beta -oxidation metabolite flux and subsequently contributes to the low rate of glucose oxidation that characterizes the developing rat intestine (33, 34).

Caution is necessary when attempting to apply data from a rat model to human pathophysiology. The rat pups in this model were separated from maternal interactions, and the RMS + IGF formulas are missing other important biologically active components that might affect intestinal glucose transport and metabolism. It was for this reason that we compared our findings in the supplemented rat pups with the dam-fed rats and subsequently found similar patterns of gene expression when comparing these two groups with the rat pups fed RMS only. The IGF-initiated changes in gene expression are specific as evidenced by the lack of effect on GD mRNA levels by RMS supplementation.

In summary, the effects on blood glucose levels and intestinal mRNA levels of suckling rat pups were quantified in response to RMS supplementation by either IGF-I or IGF-II. Relative to RMS-fed rats, supplementation with either growth factor normalized serum glucose levels, increased mRNA levels of the transporters responsible for glucose transepithelial transport, and altered mRNA levels of key mitochondrial enzymes. The blood glucose levels and patterns of gene expression observed in the supplemented rat pup intestine were similar to those found in pups that were naturally reared and fed by their mothers. We speculate that enteral IGF-I and IGF-II shift the jejunum toward glucose transport and away from glucose oxidation in the immature rat pup.


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Child Health and Human Development Grants K08BD01225-01 (to R. H. Lane) and P01-HD-26013 (to B. Dvorak and A. F. Philipps), and the American Diabetes Association (to R. H. Lane).


    FOOTNOTES

* R. H. Lane and B. Dvorak contributed equally to this work.

Address for reprint requests and other correspondence: R. H. Lane, Mattel Children's Hospital @ UCLA, B2-375 MDCC, 10833 Le Conte Ave., Los Angeles, CA 90095 (E-mail: rlane{at}mednet.ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published August 29, 2002;10.1152/ajpregu.00172.2002

Received 18 March 2002; accepted in final form 13 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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