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Dietary Gangliosides Enhance In Vitro Glucose Uptake in Weanling Rats
From the * Nutrition and Metabolism Group,
Division of Gastroenterology, and the Correspondence: A. B. R. Thomson, MD, PhD, Zeidler Ledcor Center, 130 University Campus, University of Alberta, Edmonton, AB, Canada T6G 2X8. Electronic mail may be sent to alan.thomson{at}ualberta.ca. Background: The intestine adapts to environmental stimuli, such as modifications in dietary lipids. Dietary lipids modify brush border membrane (BBM) permeability and nutrient transporter activities. Gangliosides (GANG) are glycolipids present in human milk, but they are present only in low amounts in infant formula. Exogenous GANG are incorporated into cell membranes and increase their permeability. This study was undertaken to determine if feeding a 0.2% GANG-enriched diet for 2 weeks alters in vitro intestinal sugar absorption in weanling rats compared with an isocaloric control diet or diet enriched with polyunsaturated long-chain fatty acids. Methods: In vitro uptake of 34–96 mm glucose and fructose and morphological measurements were assessed on intestinal tissue of weanling rats. Western blotting, immunohistochemistry, Northern blotting, and reverse transcription–polymerase chain reaction were performed to determine the mRNA and protein abundance of the sugar transporters SGLT-1, GLUT2 and GLUT5. Results: Feeding GANG did not alter the rates of animal weight gain or intestinal morphology. GANG did not affect fructose uptake. Depending on the concentration of glucose, GANG increased jejunal uptake of higher concentrations of glucose by approximately 20%–60%. There were no changes in GLUT5 or GLUT2 protein or mRNA abundance. Similarly, there were no changes in SGLT-1 mRNA and protein abundance, as determined by Northern and Western blotting. However, using immunohistochemistry, SGLT-1 was lower in GANG than in controls. Conclusions: The results of this study suggest that the enhanced uptake of glucose that results from feeding 0.2% GANG for 2 weeks to weanling rats may be regulated posttranslationally. Clearly any adjustment of the content of GANG in infant formula must be studied carefully. Adequate nutrients are essential to the growth and development of an infant. Formulating an infant feeding regimen with nutrients that are similar to that of human milk is important to the well-being of infants. Gangliosides (GANG; sialic acid– containing glycosphingolipids) are natural components of all mammalian cells. Human milk contains GANG, but these are present in low amounts in infant formulas. The pattern and concentration of GANG are speciesas well as tissue-specific and are also age dependent.1 GANG are mediators for cell-to-cell, cell-to-microbe, or cell-to-molecule (toxins, hormones) interactions as antigens, receptors, and ligands,2 and also are immune modulators.3 GANG exist in clusters in the cell plasma membrane, forming domains where signal transduction molecules are abundant.4 GANG are involved in modulation of transmembrane signaling activities.5 An enrichment of GANG in the caveolae or lipid rafts may alter the production or release of proteins that may signal the adaptive process. The intestinal brush border membrane (BBM) contains approximately 20% glycosphingolipids in its lipid.6 The dominant GANG is GM3,1 which in the rat is 7 times more concentrated in neonatal than in adult intestine.7 This tissue- and age-specific composition may contribute to the intestinal adaptation to diet that alters nutrient absorption. Human milk GANG, GM1, and GM3 inhibit the adhesion of enterotoxigenic Escherichia coli to Caco-2 cells, acting as a physiologic component to protect infants against intestinal infections.8 Preterm newborn infants fed GANG-supplemented formula at a concentration of 1.43 mg/100 kcal had significantly lower fecal E coli and increased bifidobacterial counts.9 Feeding GANG to animals increases the quantity of GANG in the intestinal mucosa, brain, and blood plasma.10 The GANG GM1, GD1a, and GT1b increase the passive permeability of the membrane, as measured using unilamellar vesicles and enzymatic, as well as fluorescence-spectroscopy, techniques.11 SGLT-1, the BBM sodium-dependent glucose transporter, mediates the uptake of glucose and galactose across the BBM.12 SGLT-1 is powered by the electrochemical gradient that is generated by Na+/K+-ATPase located at the basolateral membrane (BLM).13 GLUT5, a sodium-independent facilitative transporter at the BBM, mediates fructose uptake.14 At the BLM, another facilitative transporter, GLUT2, is responsible for the transport of glucose, galactose, and fructose from the cytosol into the plasma.15 In addition to its role at the BLM, GLUT2 is also expressed in the BBM in the presence of a high luminal glucose concentration16 and may thereby contribute to sugar uptake under these conditions. The intestine adapts in response to internal and external environmental changes.17,18 This adaptation process modifies BBM fluidity and permeability, as well as carrier-mediated transport. Dietary modifications induce intestinal adaptation and nutrient absorption. For example, increasing the luminal carbohydrate content increases the abundance of hexose transporters, increasing sugar transport.19 Dietary polyunsaturated fatty acids (PUFA) and saturated fatty acids (SFA) decrease and increase the saturation of the BBM phospholipids, respectively, thereby influencing BBM lipid composition.20 Animals fed a PUFA-enriched diet have lower glucose absorption than those fed an SFA-enriched diet.18 It is possible that GANG induce an intestinal adaptive response similar to that of other lipids such as PUFA and SFA. Accordingly, the objective of this study was to investigate the effect of feeding a GANG-enriched diet on intestinal sugar uptake. It was hypothesized that feeding GANG would increase intestinal sugar uptake by altering the abundance or expression of enterocyte transporters.
Animals The principles for the care and use of laboratory animals, approved by the Canadian Council on Animal Care and the Council of the American Physiologic Society, were observed in the conduct of this study. Male Sprague-Dawley rats, averaging 42 g and 17–18 days of age, were obtained from the University of Alberta Vivarium. Animals were housed in pairs at 21°C, with 12 hours of light and 12 hours of darkness. Animals were weaned and fed experimental diets ad libitum.
The rats were randomly divided into 3 groups (n = 8). Animals were fed for
2 weeks with 1 of 3 semisynthetic isocaloric
diets21 containing
20% (wt/wt) fat (Table I). The
fat composition of the control diet was similar to that of a conventional
infant formula, with a ratio of 18:2
Probe and Marker Compounds for Sugar Uptake (3H)-inulin was used as a nonabsorbable marker to correct for the adherent mucosal fluid volume. Probes were shown by the manufacturer to be >99% pure by high-performance liquid chromatography.
Tissue Preparation for Uptake The intestine was everted and cut into rings (2–4 mm). These rings were equilibrated in preincubation beakers containing oxygenated (O2:CO2, 95:5 by volume) Krebs's buffer (pH 7.2) at 37°C for 5 minutes. Uptake was initiated by the timed transfer of the rings from the preincubation buffer to a vial containing (3H)-inulin and 14C-labeled hexose in oxygenated Kreb's buffer that had been equilibrated to 37°C in a shaking water bath.22
Determination of Uptake Rates The sugar uptake rates were expressed as nmol of substrate absorbed per 100 mg dry weight of the mucosa per minute (nmol 100 mg mucosal tissue/minute, respectively).
Tissue Preparation for RNA and Protein Analysis
Morphological Measurements
Messenger RNA Abundance
Reverse Transcription–Polymerase Chain Reaction (RT-PCR) A 10-µL aliquot of the RT reaction was amplified using a DNA thermal cycler (PTC-100 Programmable Thermal Controller, version 7.0; MJ Research Inc, Watertown, MA). The reaction contained 1.25 U Taq DNA polymerase (Gibco BRL) in a 50-µL reaction containing 2.25 mmol/L MgCl, 200 µmol/L dNTPs, 20 mmol/L Tris-HCl (pH 8.0), 50 mmol/L KCl, and 300 nmol/L of each primer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR products were separated on a 1% (wt/vol) agarose gel. The abundance of DNA was determined using laser densitometry (Model GS-670 Imaging Densitometer; Biorad Laboratories Ltd, Mississauga, Ontario, Canada).
Protein Analysis
Immunohistochemistry
Expression of Results and Statistical Analysis
Characteristics of Animals After 2 weeks of feeding, there were no differences in the animals' body weight or food intake (data not shown). In the jejunum, there were no differences in the total intestinal weight (control = 5.3 ± 0.5 mg/cm, PUFA = 5.5 ± 0.6 mg/cm, GANG = 5.8 ± 0.3 mg/cm), mucosal weight (control = 3.3 ± 0.5 mg/cm, PUFA = 4.1 ± 0.7 mg/cm, GANG = 3.5 ± 0.5 mg/cm), weight of the remaining intestinal wall, or the percentage of the intestinal wall composed of mucosa (data not shown). In contrast, in the ileum, the mean of the total intestinal weights was greater (p < .05) in GANG than in control (control = 4.3 ± 0.2 mg/cm vs GANG = 6.3 ± 0.8 mg/cm). Ileal mucosal weight was increased by GANG but this was not statistically significant (control = 2.7 ± 0.4 vs GANG = 4.8 ± 1.0; p = .124). In the jejunum, there was no significant difference among the 3 groups in the villous height, midwidth, and base width (data not shown). The crypt depth (36.6 ± 3.5, 52.3 ± 2.7, 40.2 ± 2.1 µm, respectively), was greater (p < .05) in PUFA than in controls. There were no differences in the ileum of animals on PUFA, control, or GANG (49.3 ± 5.6, 52.4 ± 4.4, 44.1 ± 3.2, respectively).
Uptake of Fructose
Uptake of Glucose
Because some of the data (Table II) suggested that GANG might influence the uptake of higher concentrations of glucose, a second series of uptake studies was performed using D-glucose in concentrations of 32, 64, 96, and 128 mmol/L and with feeding either control or GANG. Because of the high glucose concentrations, D-mannitol was added to maintain isotonicity. The jejunal uptake of 32, 64, and 96 mmol/L glucose was significantly increased (p < .05) in GANG compared with control (Table III). When the GANG content was increased to 0.04%, the jejunal uptake of only 96 mmol/L glucose was increased (Table III). In the ileum, the uptake of 32 mmol/L glucose was increased by 0.02% GANG. No changes were seen in the ileum of animals fed 0.04% GANG.
Western Blotting: Transporter Protein Abundance
Northern Blotting and RT-PCR: Transporter mRNA Expression SGLT-1 mRNA abundance was not significantly different between the treatment groups (Figure 3A). Similarly, GLUT2 and Na+/K+-ATPase  1 and β1 mRNA
abundance was similar in all groups (data not shown). GLUT5 mRNA, as
determined by RT-PCR, was also similar in all groups (data not shown).
Immunohistochemistry
Although the animals' body weights did not change with the 3 diets, the weight of the ileum was higher in GANG than in PUFA or control (data not shown). For this reason, the rates of uptake had to be expressed according to the weight of the mucosa (Jm). There was no change in intestinal morphology with diet, except for the lower crypt depth in control than in PUFA (data not shown). For this reason, uptake was not expressed according to villous surface area. There was no difference in fructose uptake in the jejunum or ileum (data not shown). This paralleled the lack of effect of diet treatment on the abundance of GLUT5 protein or mRNA, clearly demonstrating the lack of effect of these diets on intestinal fructose absorption.
There was a curvilinear relationship between glucose concentration and uptake. The kinetics of glucose uptake are described by 2 constants, Vmax and Km. There are several methods to estimate Vmax and Km, including a best fit curve analysis (Sigma Plot) and many different linear transformations of the Michaelis Menten equation. Although there are pros and cons of each of these methods,29–31 neither Sigamplot nor the Lineweaver-Burke plot showed a change in the values of the Vmaxs for glucose uptake in the jejunum or ileum of rats fed PUFA or GANG, compared with controls (Table II). The Km was increased by GANG and PUFA in the jejunum but was decreased by GANG in the ileum. The value of the apparent Km may be influenced by the effective resistance of the unstirred water layer (UWL),29,31,32 but the lack of differences in the uptake of lauric acid in studies similar to these33 suggests that the diet-associated changes in the Km were not due to differences in UWL resistance.34 Of interest, in most models of intestinal adaptation, it is the Vmax that is modified.35 The alterations in Km may reflect changes in the affinity of the transporters for their substrates, although it is unclear why there were diet-associated changes in the jejunum and not in the ileum. This altered affinity in the jejunum with PUFA or GANG could account for changes in the transport of low concentrations of glucose in the absence of variations in transporter abundance.
Because there was a suggestion from the Sigmaplot and the Lineweaver Burke plots that jejunal glucose uptake was higher in GANG than in control, when using glucose concentrations of 4–64 mmol/L (Table II), we undertook a second series of experiments to explore possible enhancing effects of GANG on glucose uptake, using higher glucose concentrations (32–128 mmol/L). Glucose uptake was greater at 32, 64, and 96 mmol/L in GANG than in control (Table III). We do not have an explanation of why 0.02% GANG did not affect the jejunal uptake of 128 mmol/L glucose (Table III). The enhanced glucose uptake in animals fed GANG vs control was expected to be associated with increased SGLT-1 abundance. However, as measured by Western blotting, there was no difference in the jejunum, and there was a decline in the ileum (Figure 1). The abundance of SGLT-1 was also assessed by immunohistochemistry, and again GANG did not increase the abundance of SGLT-1; indeed, SGLT-1 was lower in GANG than in control (Figures 2 and 3B). Also, the altered uptake of glucose in animals fed GANG was not due to a change in distribution of transporters along the villus (data not shown). In addition, there was no change in SGLT-1 mRNA abundance in response to dietary manipulation (Figure 3A). Furthermore, dietary lipid changes had no effect on the abundance of GLUT2 or Na+/K+-ATPase proteins or mRNAs in the BLM (data not shown). The PKC signaling pathway modulates SGLT-1 posttranslationally via regulating exocytosis of SGLT-1 from the cytosol to the BBM.36 We did not measure SGLT-1 phosphorylation or BBM lipid composition or fluidity, so we cannot comment if these may have also played a role in the enhanced uptake of higher concentrations of glucose in rats fed GANG. We also did not determine where the SGLT-1 is located in the BBM, so that a further posttranslational mechanism could include the positioning of SGLT-1 in a membrane domain optimizing transporter activities, such as BBM lipid rafts. The activity of SGLT-1 may be enhanced by an increase in Na+/K+-ATPase activity at the BLM. It is possible that incorporation of GANG in the BLM increases the Na+/K+-ATPase activity via signal transduction modulation, thereby enhancing the activities of SGLT-1. Our data did not show any change in the abundance of Na+/K+-ATPase (data not shown), but it is possible that a change in activity occurs in the absence of an alteration in protein abundance. The enhanced glucose uptake may be due to an increase in the total GANG and GD3 content in the intestinal mucosa and decreased GM3 content,10 which reflects the dietary GANG that were fed to the animals. It was shown that GD3 was mostly localized at the BLM, and only a small amount is found in the BBM. Introducing more sialic acid from GM3 to GD3 may increase glucose uptake without apparently changing the number of SGLT-1 transporters. This raises the possibility that a specific dietary GANG or concentration of GANG may be required for nutrient uptake in young animals. GANG are enriched in lipid rafts/caveolae.37 Runembert and colleagues38 demonstrated that SGLT-1 is localized to microdomains of the BBM, and it might reach its optimal activity in a low fluidity environment. Inhibition of Na+-dependent glucose absorption by the binding of cholera toxin or viral envelope glycoprotein (HIV-1 gp 120) occurs in the glycosphingolipid-enriched microdomains.39 Park and colleagues10 have provided evidence that there is an interaction between GANG and phospholipid polyunsaturates, as well as cholesterol. We speculate that GANG altered glucose uptake by way of modification in the lipid composition of the BBM or microdomains of the BBM, reducing the jejunal uptake of low concentrations of glucose by decreasing the affinity (increasing the value of the Km) of the transporters for glucose (Table II), and enhanced the jejunal uptake of 32, 64, and 96 mmol/L glucose (Table III) by increasing the activity of SGLT-1 in a posttranslational manner. The 0.02% GANG dose in the diet was chosen to reflect the concentration of GANG observed in human milk,40 and this is a "physiologic" rather than a pharmacologic dose. Clearly, there was less effect of 0.04% GANG on glucose uptake (Table III). This suggests that an excessively high dietary concentration of GANG fails to stimulate the uptake of higher concentrations of glucose, and any proposed adjustment of the content of GANG in infant formula must be studied carefully. In addition, the mechanisms of this enhancement in glucose uptake within what appears to be a narrow range of dietary GANG concentrations must be established, and the clinical significance of infant formula enrichment with GANG needs to be assessed. The authors would like to thank Ms. Celina J. Birecki, MSc, formerly of the Nutrition and Metabolism Group, Division of Gastroenterology at the University of Alberta, for her contributions to this paper. Received for publication May 16, 2006. Accepted for publication April 12, 2007.
Journal of Parenteral and Enteral Nutrition, Vol. 31, No. 5,
423-429 (2007)
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MATERIALS AND METHODS
RESULTS
-2 to 18:3
.05. 
