1Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi; 2Department of Pathology, The University of Chicago, Chicago, Illinios; and 3Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, Mississippi
Submitted 23 February 2005 ; accepted in final form 12 July 2005
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ABSTRACT |
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sodium-dependent glucose transporter 1; adaptation; regulation; nongenomic
Historically, transport physiologists interested in the adaptive regulation of nutrient uptake by the brush border membrane (BBM) of enterocytes have focused on how diet, development, and disease states trigger selective gene expression, leading to changes in the abundances of specific transporters. These genomic responses are known to occur over hours to days, during the life history of individuals, and during the evolution of species. Despite the importance of genomic mechanisms of adaptation, the responses are not fast enough for animals to modulate the absorptive functions of the BBM during the processing of individual meals. Moreover, the energetic costs associated with the synthesis of additional transporters for the processing of individual meals would be high.
It has been established that carrier-mediated glucose uptake can be modulated in <60 min, a time scale that is consistent with the processing of individual meals (7, 18, 25). The ability to rapidly regulate nutrient transporters provides an adaptive mechanism that allows absorptive functions to be matched to existing lumenal nutrient concentrations and thereby complements the slower genomic responses to diet composition. Moreover, based on the rapid and pronounced regulation of ion secretion (4, 43), the magnitude of rapid adaptation for the nutrient transporters could exceed what is possible by genomic responses alone. Determining the role of nongenomic regulation in adaptive modulation of transport functions is dependent on measuring changes in absorption that occur in <10 min to avoid the participation of genomic mechanisms. Unfortunately, most studies of rapid regulation have used longer periods of time (e.g., 30 and 60 min), and this may be sufficiently long for the production of new proteins.
A key component of a rapid response is the ability of enterocytes to recognize and respond to changes in lumenal nutrients and to host signaling molecules (38, 44, 51). Corresponding with this, carrier-mediated glucose uptake is higher 30 min after activation of a "glucose sensor" (11). Conversely, enterocyte absorptive functions are lower after an exposure to inflammatory cytokines (14, 29), although the responses are not universal (17). The regulatory influence of adenosine on the intestine is of basic interest and clinical importance. Adenosine is a conditionally essential nutrient for the intestine (34) but also functions as an ubiquitous host signaling molecule that regulates the intestinal functions of ion secretion, motility, and immunity (6, 13, 15, 23, 28, 39, 42, 47, 48). Of particular importance is the participation of adenosine in mediating inflammatory responses at sites of tissue injury.
Despite the multiple roles of adenosine in regulating intestinal health and functions, the influence of adenosine on intestinal absorptive functions is uncertain. Therefore, the role of adenosine as a lumenal signaling molecule that regulates carrier-mediated glucose transport by the small intestine was established using three complementary experimental approaches. The first two involved measuring rates of glucose transport by the intact mouse small intestine after chronic (7 days) and acute (from 2 to 10 min) exposure to adenosine and a disodium salt of AMP that is stable at room temperature. The third evaluated the clinical relevance of responses to adenosine using mice to measure in vivo absorption of an oral dose of 3-O-methylglucose (3-OMG) in the presence and absence of AMP.
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MATERIALS AND METHODS |
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All aspects of the research using animals were approved by the Mississippi State University Institutional Animal Care and Use Committee and were performed in facilities accredited by the American Association for the Accreditation of Laboratory Animal Care. Adult mice (620 wk of age) of both sexes and of the Swiss-Webster and BALB/c strains were obtained from a commercial supplier (Taconic; Germantown, NY) or were produced by a breeding colony maintained at Mississippi State University. The mice were housed as 5 mice/cage in a controlled environment (12:12-h light-dark cycle, 22 ± 2°C, and 50% relative humidity) and were allowed free access to tap water and a rodent chow (Lab Diet 5001, PMI Feed; St. Louis, MO).
Measurement of Intact Tissue Glucose Transport
The everted sleeve method (24) was used as a sensitive functional assay to detect changes in nutrient transport in response to adenosine and AMP (mol wt = 391.2). Glucose was selected as the model nutrient because it is possible to measure the carrier-mediated component of uptake, because there are selective inhibitors for the apical membrane glucose transporters SGLT1 and GLUT2, and because apical membrane glucose uptake can be rapidly modulated by altering the densities and proportions of SGLT1 and GLUT2 (26).
Mice were killed by CO2 asphyxiation for harvest of the small intestine. In light of the declining proximal-to-distal gradient for glucose uptake, the majority of studies measured the responses of the proximal small intestine (beginning 5 cm distal to the pyloric sphincter). For studies searching for regional differences in responses, additional tissues were prepared from the distal small intestine (extending 5 cm proximal to the ileocolonic junction). From each region, four to eight sleeves of 1-cm length were secured by silk ligatures onto stainless steel rods that approximated sleeve diameter (1.5, 2, and 3 mm). The sleeves were kept in cold (24°C), aerated (95% O2-5% CO2) Ringer solution during and after the mounting preparation. Measurements of absorption began 45 min after collection of the small intestine by exposing the sleeves to 37°C mammalian Ringer solution with and without adenosine and AMP and using different conditions [length of exposure, concentrations of adenosine and AMP, presence or absence of inhibitors for SGLT1 (phloridzin) and GLUT2 (phloretin), and adenosine receptor antagonists]. After the exposure, tissues were suspended for 2 min in an uptake solution consisting of Ringer solution that was aerated and stirred (1,200 rpm) and contained tracer concentration (2 µM) of D-[14C]glucose with and without 50 mM unlabeled D-glucose. The tracer L-[3H]glucose was added to the uptake solution for correction of D-glucose in the extracellular fluid and was passively absorbed. After a 2-min incubation, tissues were rinsed for 20 s in cold Ringer solution, carefully blotted to remove adherent fluid, removed from the rods, and placed in tared vials. After the wet mass was recorded, the tissues were dissolved (Solvable, Perkin-Elmer; Boston, MA), scintillant solution was added (Ultima Gold, Perkin-Elmer), and the associated radioactivity was quantified by liquid scintillation counting. Rates of glucose uptake (in nmol or pmol/min) were calculated and normalized to wet tissue mass and, due to the use of L-glucose, represent the carrier-mediated component of absorption.
Chronic Exposure of Mice to AMP
Mice of the BALB/c and Swiss-Webster strains were individually housed and continued to be fed the rodent chow. Each day for 7 days, the mice were provided in the morning (08000900 h) a supplement consisting of canned sweet potato (vehicle, 500 mg/mouse) with and without 100 mg AMP (0.26 mmol). After the 7-day period of supplementation, rates of glucose transport by intact tissues from the proximal and distal small intestine were measured with 50 mM glucose in the incubation solution.
Acute Responses of Intact Mouse Tissues
Tissues from the proximal small intestine of BALB/c mice fed the commercial diet were used to study the accumulation of glucose at tracer concentration (2 µM) after exposure to 5 mM adenosine and AMP for varying periods (010 min). This was accomplished by exposing the tissues to 37°C aerated Ringer solution and switching to Ringers solution with adenosine or AMP such that the combined length of exposure was 10 min for all tissues. Preliminary studies showed that tissue uptake was stable up to 10 min of exposure, but, after 15 min, rates of uptake began to decline. Phloridzin and phloretin were added to the uptake solution (0.5 mM) to determine whether the response to AMP involved SGLT1 and GLUT2, respectively.
Subsequent studies measured glucose uptake after tissues were exposed for 7 min to different concentrations of adenosine and AMP (050 mM). Osmolarity of the solutions was maintained constant by isosmotic replacement of the NaCl fraction in the Ringer solution.
Responses to Adenosine Receptor Antagonists
The possible involvement of an adenosine receptor in mediating the responses was examined using intact tissues from the proximal small intestine that were exposed for 7 min to Ringer solution with adenosine (5 mM) in the presence or absence of theophylline (100 µM), which is a nonselective antagonist of adenosine receptors, before the uptake of tracer D-glucose (2 µM) was measured. Subsequent experiments sought to identify the role of specific adenosine receptors in regulating the uptake of tracer D-glucose by exposing tissues to Ringer solution with adenosine (5 mM) in the presence and absence of antagonists for the A1 receptor (8-phenyltheophylline; 0.1 µM) and A2 receptors [3,7-dimethyl-1-propargylxanthine (DMPX); 0.1 µM].
Activation of adenosine receptors expressed on plasma membranes can alter adenylyl cyclase (AC) activity and, hence, intracellular levels of cAMP. Therefore, to determine whether the responses to adenosine were mediated by a signaling pathway that increases intracellular cAMP, rates of glucose uptake were compared among tissues from the proximal small intestine that were exposed for 7 min to Ringer solution with and without 5 mM adenosine and in the presence and absence of forskolin (10 µM), which was used to increase cellular cAMP levels.
Absorption of Orally Administered 3-OMG
To understand whether the responses of intact tissues are of clinical relevance, young adult female BALB/c mice were orally dosed with 3-OMG, a nonmetabolizable glucose analog, which is a substrate for SGLT1 and other glucose transporters. Before the 3-OMG tolerance test, mice were trained over a 3-day period to rapidly consume (in 23 min) a vehicle (small, moistened balls prepared with 0.2 g wheat flour, 0.1 g powdered cheese, and 0.1 g water). It was decided to use a vehicle for the 3-OMG challenge instead of oral gavage to avoid stressing the mice, which may cause the release of glucocorticoids and thereby decrease the magnitude of nongenomic responses (18, 37). On the day of the study, 3-OMG (100 µmol of both unlabeled and tracer 3H labeled) was added to the dough balls with and without 26 µmol (10 mg) AMP. After consuming the vehicle with 3-OMG, mice were killed at 10, 15, 30, and 60 min (n = 910 for each time and treatment) by CO2 asphyxiation, and blood samples were immediately withdrawn from the inferior vena cava. The liver was collected, the small intestine was removed (from the pyloric sphincter to the ileocolonic junction), and the contents were flushed out using cold Ringer solution. The samples of whole blood, the entire small intestine, and a portion of the liver (0.51 g) were oxidized (model 307, Packard Bioscience; Meriden, CT). The 3-[3H]OMG was recovered in Permafluor E (Perkin-Elmer), and radioactivity was quantified by liquid scintillation counting. Samples of the dough ball were analyzed to determine radioactivity per nanomole of 3-OMG, and this was used to quantify 3-OMG in the samples of blood (nmol/ml) and tissue (nmol/g).
Chemicals
Perkin-Elmer supplied the D-[14C(U)]-glucose and L-[1-3H(N)]-glucose, and the 3-O-([3H]methyl)-D-glucose was purchased from Sigma Chemical (St. Louis, MO). The stable form of AMP was generously provided by Otsuka Pharmaceutical. Adenosine and all other chemicals were purchased from Acros Organics (Fairlawn, NJ) and Sigma Chemical and were of the highest purity available.
Statistical Analysis
Rates of nutrient uptake are reported as means ± SE. Data for the chronic exposure study were analyzed by two-way ANOVA to search for the main effects of treatment, strain, gender, and region, with specific differences identified using an unpaired Student's t-test. Data for the uptake responses of tissues from BALB/c mice after acute exposures were analyzed by one-way ANOVA to search for differences among incubation times, concentrations of adenosine and AMP, and the treatments with adenosine in the presence and absence of pharmacological agents (phloridzin, phloretin, theophylline, 8-phenyltheophylline, DMPX, and forskolin). When a significant main effect was detected, specific differences were identified by Duncan's test. An unpaired Student's t-test was used to determine whether the vehicle with AMP increased in vivo accumulation of 3-OMG in the blood, small intestine, and liver after oral administration. For all analyses, a value of P < 0.05 was considered as significant. All statistical analyses were performed using the Statistical Analysis System (version 8.2, SAS Institute; Cary, NC).
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RESULTS |
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Chronic exposure to AMP. Strain and gender effects were not detected for the response to chronic dietary exposure to AMP. Therefore, values for both sexes of Swiss-Webster and BALB/c strains of mice were pooled for statistical analysis.
The daily supplement of 256 µmol AMP for 1 wk did not result in differences in body mass (control: 34.9 ± 1.8 g; AMP: 34.9 ± 1.4 g) and intestinal length (control: 47.1 ± 1.0 cm; AMP: 46.9 ± 1.4 cm) and mass (control: 2.3 ± 0.1 g; AMP: 2.3 ± 0.1 g). Rates of glucose uptake measured at 50 mM declined from the proximal to distal small intestine for control mice and those receiving the AMP supplement, with rates of uptake in the proximal small intestine higher compared with the distal region (Fig. 1). However, mice receiving the AMP supplement had higher rates of glucose uptake in the distal small intestine compared with control mice (P < 0.05), with a nonsignificant treatment difference for the proximal intestine (P = 0.11).
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The concentration of 3-OMG in the blood of mice fed 3-OMG alone increased to a maximum at 30 min with similar concentrations measured at 60 min (Fig. 10). Mice that consumed the vehicle with the combination of 3-OMG and AMP had higher concentrations of 3-OMG in whole blood collected at 10 min compared with mice receiving the vehicle with 3-OMG alone, with the difference significant after 15 min. Differences were not detected at later times. There was a corresponding higher concentration of 3-OMG after 15 min in the liver of mice fed the vehicle with AMP (P = 0.06) but not at other times (data not shown). Similarly, accumulation of 3-OMG in the entire small intestine tended to be higher at 15 min (P = 0.09) for mice that consumed the vehicle with AMP.
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DISCUSSION |
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The rapid increase in glucose uptake after the exposure to adenosine and the more rapid accumulation of 3-OMG in small intestine tissue, blood, and the liver of mice that received AMP is also pertinent to the controversy surrounding the higher absorptive capacities measured in vivo compared with those calculated from in vitro rates (30). The present results, and the reports cited above, indicate that dietary and host signaling molecules elicit higher rates of uptake during the processing of meals. However, in vitro studies historically have used unstimulated tissues, which would be transporting glucose at basal, not maximum, rates. In contrast, the infusion of high glucose concentrations into the intestine for in vivo measurements would trigger a nongenomic increase in uptake, resulting in higher transport capacities.
Adenosine, Adenosine Receptors, and Signaling Pathways
Adenosine is somewhat unusual among the regulatory signals that are known to modulate enterocyte functions in that it can originate from two sources: the diet and endogenous production. Although data are not available for dietary intake of adenosine, an estimate can be provided based on information from several sources. Intracellular concentrations of ATP range from 210 mM, with lower amounts of ADP and AMP (33). Additional sources of dietary adenosine include cellular DNA, RNA, and NAD, which are hydrolyzed lumenally by pancreatic enzymes, with adenosine released at the BBM by 5'-ectonucleotidase activity (16). Another consideration is that the daily intake of purines by United States adults is 6001,000 mg/day (35). Because the amount of adenine in a cell is greatly exceeded by adenosine, adenine intake underestimates the daily intake of adenosine. Collectively, these findings suggest 5 mM is a conservative estimate for the concentration of adenosine that would be present in the lumen of the intestine of individuals eating the Western human diet. The second source is endogenous production of adenosine and associated nucleotides (AMP, ADP, and ATP), which are secreted to regulate a diversity of intestinal characteristics and functions. This includes secretion at sites of tissue injury to modulate inflammatory responses (pro- or anti-inflammatory) and the secretion by lumenal neutrophils in response to pathogens (39). Host production of adenosine can be substantial, particularly when secreted adenine nucleotides are included (8). Collectively, these findings indicate that a lumenal concentration of 1 mM adenosine, which elicited the maximum increase in glucose uptake by intact tissues, is physiologically relevant.
The intestinal mucosa expresses four types of adenosine receptors, A1, A2A, A2B, and A3, yet little is known about enterocyte expression of adenosine receptors and the potential roles in regulating nutrient absorption and probably other enterocyte functions. The ability to block the response of the everted sleeves to adenosine with theophylline indicates that the BBM of the small intestine includes receptors for adenosine. The subsequent use of two receptor-specific antagonists suggests taht one or more of the adenosine A2 receptors, but not the A1 receptor, is responsible for the responses of the BBM of the mouse small intestine, but we do not yet know the identity of the specific type of A2 receptor.
Adenosine receptors are coupled to G protein-mediated signaling pathways (13), with the specific coupling of G proteins and signaling pathways varying among the four receptor types and among tissues. A1 and A3 receptors are usually coupled with Gi proteins that inhibit AC, whereas the A2A and A2B receptors are coupled with Gs proteins that stimulate AC. Activating AC using forskolin caused an 80% increase in glucose uptake by intact tissues (present study), which is consistent with forskolin-induced increases in the densities of SGLT1 transporters in the BBM (49) and higher rates of carrier-mediated uptake of glucose, bile acids, and amino acids (31). The response to forskolin in conjunction with the lack of a response to adenosine in the presence of an antagonist of A2 receptors implicates the involvement of at least one of the A2 receptors.
Although the downstream signaling events after cAMP were not determined, the roles of PKA and PKC in regulating glucose uptake have been established using oocytes (50), COS-7 cells (46), perfused segments of the intestine (9, 26), and isolated intact tissues (2). However, the responses to PKA and PKC are not consistent among nutrient transporters and cell types. For example, activating PKA increases glucose uptake by small intestine tissue (2) and oocytes expressing SGLT1 (50), whereas activation of PKC causes a decrease. In contrast, activation of PKC elicits an increase in glucose absorption via GLUT2 (26). The contrasting responses indicate that regulation of nutrient transport systems by adenosine is complex and involves multiple signaling pathways.
Mechanisms for the Increase in Glucose Transport
The majority of studies reporting rapid responses for SGLT1 and other nutrient transporters have used exposure periods of 3060 min. This is sufficiently long to allow for at least partial genomic responses. The higher rates of glucose transport measured in <5 min after an exposure to adenosine is too rapid to be explained by genomic mechanisms, which would require the synthesis and insertion of functional transporters into the BBM. Hence, nongenomic mechanisms must play a central role in the rapid adaptation of glucose uptake.
In unstimulated intestinal tissue, glucose uptake is by a "basal" density of SGLT1 that is maintained in the BBM (18), with the majority of BBM glucose transporters (SGLT1 and GLUT2) associated with an intracellular fraction. The rapid increase in tracer glucose uptake is consistent with the trafficking of glucose transporters from an intracellular site to the BBM (27).
Moreover, the virtual elimination of BBM tracer glucose uptake when control and adenosine-stimulated tissues were exposed to phloridzin suggests that SGLT1 is responsible for the majority of basal glucose uptake and the increase in response to adenosine. Corresponding with this, exposing the intact rat intestine to polyamines (45) induces rapid and simultaneous increases in glucose uptake (apparent by 5 min) and the abundance of SGLT1 in the BBM (measured at 15 min). It is possible there was a simultaneous upregulation of GLUT2 in conjunction with SGLT1, as previously reported (18, 26). Because GLUT2 is a lower-affinity transporter, it is difficult to detect changes in rates of uptake based on the accumulation of tracer concentrations of labeled glucose via GLUT2. Still, including phloretin in the incubation solution decreased the tissue accumulation of tracer D-glucose by control tissues and those exposed to AMP.
An alternative mechanism is that transporter site density in the BBM may not change in response to adenosine. Instead, the activity of transporters in the BBM could be regulated. Although there is less direct evidence for the activation/deactivation of BBM transporters, the "in situ" regulation of transporters in the BBM by phosphorylation remains a distinct possibility (21). SGLT1 includes several potential sites for phosphorylation by PKA and PKC (20), and there is evidence SGLT1 activity may be regulated by interactions with other intracellular proteins (e.g., heat shock proteins) (19).
A New Paradigm of Transporter Regulation
Nongenomic regulation has not been adequately recognized as a mechanism for the adaption of nutrient transport. Specifically, the 70% or more increase in carrier-mediated glucose uptake by the intact mouse small intestine after a 7-min exposure to adenosine or AMP is comparable in magnitude to the slower genomic response that occurs over several days when mice are switched between diets with 0% and 55% sugar (24). Hence, adaptative regulation of nutrient transport includes nongenomic responses that rapidly (<5 min) modulate the densities or activities of transporters in the BBM, whereas genomic responses establish the total cellular abundance of transporters that are available for rapid regulation.
Perspectives
Of particular importance is learning how adenosine can be prosecretory and proabsorptive. This will require a better understanding of the types, proportions, and distributions of adenosine receptors expressed by enterocytes and the associated signaling pathways. These efforts will be complicated by the differential targeting of the receptor types to the apical and basolateral membrane domains of polarized cells (3, 22, 40, 48), the trafficking of receptors to the apical membrane after exposure to adenosine (48), and how the proportions of the different receptors in the membrane domains are responsive to health status (28, 42).
There is also a need to understand whether the response to adenosine is unique to SGLT1 or if it is shared by transporters for other nutrients, such as amino acids and fatty acids. Learning which transporters are responsive to adenosine, and other signaling molecules, will assist in identifying nutrients that are most likely to become limiting during disease states as well as the development of therapeutic strategies to reduce the incidence and severity of malabsorption.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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