Symposium: Glucagon-Like Peptide 2: Function and Clinical Applications

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1 Symposium: Glucagon-Like Peptide 2: Function and Clinical Applications Glucagon-Like Peptide 2: A Key Link between Nutrition and Intestinal Adaptation in Neonates? 1,2 Douglas Burrin,* 3 Xinfu Guan,* Barbara Stoll,* Yvette M. Petersen and Per T. Sangild *U.S. Department of Agriculture/ARS Children s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, and Division of Animal Nutrition, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Copenhagen, Denmark ABSTRACT This paper reviews the evidence from recent studies in young piglets to examine the hypothesis that glucagon-like peptide 2 (GLP-2) is a physiologically relevant hormonal signal linked to the intestinal adaptation associated with enteral nutrition in neonates. Observations that support the hypothesis include, 1) the GLP-2 secretory response to enteral nutrition is functional as early as late gestation, 2) parallel changes in intestinal growth and circulating GLP-2 occur in response to the quantity and composition of enteral nutrition after birth, and 3) the acute temporal changes in intestinal metabolism and circulating GLP-2 concentrations in response to enteral nutrition are generally coincident. In contrast, however, the lack of intestinal trophic responses to both pharmacological GLP-2 concentrations in the fetus and weanling pigs, and to physiological GLP-2 concentrations in neonates raises doubts concerning the physiological relevance of GLP-2 as a enterally mediated trophic signal. A more definitive test of this hypothesis will require further studies that assess the intestinal metabolic response to enteral nutrition using experimental approaches that block GLP-2 action. J. Nutr. 133: , KEY WORDS: cell proliferation apoptosis gut hormone enteral nutrition blood flow nitric oxide Glucagon-like peptide 2 (GLP-2) 4 is a member of the glucagon superfamily of peptide hormones produced mainly in the gastrointestinal tract, but also in the central and peripheral nervous system of mammals (1). GLP-2 is produced from the posttranslational processing of the proglucagon gene expressed 1 Presented at the Experimental Biology meeting, April , San Diego, CA. The symposium was sponsored by The American Society for Nutritional Sciences and supported in part by the USDA-Human Nutrition Program, NPS Pharmaceuticals, NIH-Office of Dietary Supplements and Wyeth Nutritionals. The proceedings are published as a supplement to The Journal of Nutrition. This supplement is the responsibility of the guest editors to whom the Editor of The Journal of Nutrition has delegated supervision of both technical conformity to the published regulations of The Journal of Nutrition and general oversight of the scientific merit of each article. The opinions expressed in this publication are those of the authors and are not attributable to the sponsors or the publisher, editor or editorial board of The Journal of Nutrition. Guest Editors for the symposium publication are Doug Burrin, USDA/ARS Children s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas and Kelly Tappenden, Department of Food Science and Human Nutrition/Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois. 2 This work was supported by federal funds from the U.S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number and from the National Institutes of Health Grant HD33920 (D.G.B.). The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 3 To whom correspondence should be addressed. dburrin@bcm.tmc.edu. 4 Abbreviations used: GLP-2, glucagon-like peptide 2; NOS, nitric oxide synthase; TPN, total parenteral nutrition. in the enteroendocrine L-cell located primarily in the distal intestine and nucleus tractus solitarius region of the brain (2,3). GLP-2 is secreted into the blood as a 33-amino acid peptide in response to direct enteral nutrient stimulation of L-cells and indirect action from enteroendocrine and neural inputs, including gastric inhibitory peptide, gastrin-releasing peptide and the vagus nerve (1). The biological activity of GLP-2 in circulation is significantly influenced by the rapid N-terminus cleavage by dipeptidylpeptidase IV from its fulllength (GLP-2, 1 33) peptide form to a relatively inactive truncated form (GLP-2, 3 33). In recent years, there has been increasing interest in GLP-2 and its potential physiological role in intestinal adaptation, since the studies by Drucker and others first showed that GLP-2 is a potent intestinal trophic peptide (4). A number of subsequent studies have explored the effects of GLP-2 in various models of intestinal adaptation (2,3). The focus of this symposium was on the significance of GLP-2 in the clinical condition, short-bowel syndrome and the potential role of GLP-2 as a trophic signal between enteral nutrition and intestinal adaptation. The other contributors to this symposium highlight the historical development of GLP-2 (5), the cellular and molecular mechanisms of action of GLP-2 (6), its relationship with SCFA-mediated intestinal growth (7) and the clinical implications of GLP-2 in short-bowel patients (8). The focus of the current paper is to review a series of studies in young developing piglets in order to examine whether GLP-2 is indeed a hormonal signal that mediates at least part /03 $ American Society for Nutritional Sciences. 3712

2 NUTRITION AND NEONATAL INTESTINAL ADAPTATION 3713 of the intestinal trophic response associated with enteral nutrition. Enteral nutrition, gut growth and GLP-2 secretion In a previous study in postnatal piglets, enteral nutrition was shown to be the primary stimulus for both intestinal mucosal growth and for the secretion and increased circulating concentration of GLP-2 (9). Hence, the circulating GLP-2 concentration is significantly reduced during total parenteral nutrition (TPN) and an enteral intake of at least 40% of the total nutrient intake is necessary to increase plasma GLP-2. More importantly, several measures of intestinal growth, including tissue composition, structure and metabolic activity, are similarly decreased by TPN and increases significantly when the enteral intake reaches 40 60% of total intake. Thus, in postnatal piglets there is a strong correlation between the level of enteral intake, circulating GLP-2 concentration and intestinal growth. Using the same piglet model, we next set out to determine the relative intestinal trophic effect of the main macronutrient components in our elemental diet and investigated whether the response is correlated with plasma GLP-2 concentration. In vivo studies in adult humans indicate that carbohydrate and fat are more potent GLP-2 secretagogues than protein (10,11). To test this, we used the same elemental diet as in our previous study (9), but provided only the amino acid, carbohydrate or lipid component as the sole enteral nutrient via intragastric infusion, while providing the other two macronutrients via an intravenous infusion. We also kept the relative proportions of the three macronutrients the same; carbohydrate, amino acids and lipid were 44, 33 and 23% of the total caloric intake. We found that the trophic effects of the three macronutrients are ranked accordingly, carbohydrate amino acids lipid, based on measurements of intestinal weight as an endpoint (12). Moreover, we found that the rank order of the plasma GLP-2 concentrations is similar to intestinal weight, being greatest for carbohydrate amino acid lipid. Therefore, not only is the level of enteral intake correlated to the circulating GLP-2 concentration and intestinal growth, but enteral intake of the specific macronutrients also produces similar relative changes in plasma GLP-2 and intestinal growth. Stage of development We have also used the young piglet to examine whether stage of development affects the intestinal trophic and GLP-2 secretory response to enteral nutrition. Our interest in this question stems from our observations in pigs that the growth rate of the intestine, relative to body weight, increases markedly in late gestation and continues to increase even more rapidly just after birth (13). The neonatal intestinal growth spurt coincides with the onset of enteral nutrition (colostrum ingestion) in the suckling neonate. Our recent study demonstrated that the plasma GLP-2 concentration increases gradually during late gestation, peaks at 1 2 d after birth ( pmol/l) and is temporarily increased in enterally versus parenterally nourished term newborn piglets (14). In preterm newborn pigs, this elevated GLP-2 level in enterally versus TPN-fed piglets is delayed compared with pigs delivered at term. These studies also showed that even pig fetuses (90% gestation) respond to colostrum feeding in utero with a large increase in circulating GLP-2 level. Neither colostrum nor amniotic fluid swallowed by the fetus contains notable amounts of GLP-2, and enteral nutrition is associated with marked intestinal growth in both fetal and neonatal pigs. In contrast, increased small intestinal growth is absent in newly weaned 4-wk-old pigs despite a rise in circulating GLP-2 levels (14). Taken together, these results demonstrate that the intestinal trophic and GLP-2 endocrine secretory responses to enteral nutrition become functional in the late gestation and that such response may be involved in the rapid intestinal growth associated with the normal transition from parenteral to enteral nutrient in the neonate. In the fetus, the swallowing of amniotic fluid is known to affect rapid prenatal intestinal growth (15), but the mechanism by which this occurs is unlikely to involve GLP-2, because of the low circulating GLP-2 levels and the lack of intestinal responsiveness to pharmacological doses of GLP-2 at this time, described below (16). Intestinal responsiveness to GLP-2 dose In order to further elucidate the potential link between GLP-2 and intestinal growth, we compared the trophic responsiveness of the neonatal intestine to artificial manipulation of circulating GLP-2. To do this we used the model of the TPN-fed piglet, a condition that produces a relatively reduced intestinal growth rate and low plasma GLP-2 concentrations, and quantified the changes in intestinal growth both in response to increasing intravenous doses of GLP-2 and at different stages of development. Some of these results are compiled in Figure 1 illustrating GLP-2 effects at four different FIGURE 1 Intestinal growth (intestinal wet weight) and functional (maltase activity and glucose absorption) responses to 1 wk of GLP-2 treatment in fetal pigs (93% gestation), 1-wk-old TPN-fed preterm pigs (born at 93% gestation), 1-wk-old term TPN-fed pigs, and in weaned pigs (31 d). The average intestinal maltase activity and glucose absorption was measured in vitro. *P 0.05 versus controls. Data compiled from Buddington et al. (2000), Petersen et al. (2001, 2002) and Nielsen et al. (2003).

3 3714 SYMPOSIUM stages of development: fetuses, premature newborn pigs, term newborn pigs and weanling pigs. In our first study, we found that intravenous infusion of a pharmacological dose of GLP-2 significantly increases intestinal mucosal growth in TPN-fed premature piglets and, in effect, prevents the mucosal atrophy associated with TPN versus enteral nutrition (17). We next examined the effect of stage of development, by infusing the same dose of GLP-2 into fetuses and term TPN-fed piglets. Consistent with our first study, we found that intravenous GLP-2 infusion significantly increases intestinal mucosal growth in TPN-fed term piglets (16). In contrast, however, we found that the infusion of the same dose of GLP-2 in utero for 7 d has no effect on fetal intestinal growth or cell proliferation, although it does modify the expression and activity of some brush border enzymes. Brush border enzymes (e.g., maltase activity) are affected, although to a different extent, in both preterm and term piglets (16,18), whereas intestinal absorption of glucose is stimulated only in term TPN-fed piglets (19) (Fig. 1). Finally, in 4-wk-old pigs exhibiting weaning-induced mucosal atrophy, exogenous GLP-2 treatment does not alter either intestinal growth or function (20,21). This lack of intestinal response in both the fetal and weaned pig was observed despite 1) evidence of comparable intestinal GLP-2 receptor mrna expression in fetal, premature, term and weaned piglets and 2) the fact that we infused a pharmacological GLP-2 dose that produced supraphysiological circulating concentrations (e.g., 300 pmol/l). Collectively, these observations suggest that the prenatal increase in circulating GLP-2 in the pig plays a limited role for the intestinal growth in the fetus, and that the reported effects of exogenous GLP-2 on intestinal growth and function after birth are highly agespecific with the most pronounced effects occurring during the first few weeks postnatally. In a more recent study, we compared the intestinal trophic response of TPN-fed piglets to increasing doses of exogenous GLP-2 infusion (22). We infused human GLP-2 at three doses for 7 d producing circulating concentrations in a high physiological range ( pmol/l, low dose) and a supraphysiological range ( 350 pmol/l, medium dose and 750 pmol/l, high dose). Analysis of the results using linear regression showed that most endpoints of intestinal growth are dose-dependently increased by GLP-2. Moreover, regression analysis found that measures of intestinal mass are also highly related (R ) to circulating GLP-2 concentration. However, when we used a means comparison test, we found that the lowest GLP-2 dose (i.e., within the physiological range) has no significant effect on any measure of intestinal growth when compared to TPN alone. These findings show the remarkable responsiveness of the neonatal small intestine to pharmacological increases in the circulating GLP-2 concentration, yet they raise doubt as to whether GLP-2 has a trophic effect under physiological conditions. On the other hand, it is possible that the physiological circulating GLP-2 concentrations that we acheived in TPN-fed piglets do not reproduce the local tissue concentration, which occurs with enteral feeding pehaps due to the high activity of dipeptidylpeptidase IV enzyme. Thus, under the conditions of TPN, affecting the local tissue concentration and stimulating a trophic effect may require a higher plasma level of GLP-2 than what otherwise might occur during enteral feeding. Acute responsiveness to enteral nutrition and GLP-2 A final aspect of GLP-2 function is the postprandial temporal responsiveness of GLP-2 secretion and intestinal growth to enteral nutrition. Studies in adult humans and piglets demonstrated that GLP-2 secretion is rapidly increased within min after an enteral feeding (10,23). In addition, the intestinal mucosa is characterized by a relative high rate of metabolism and cellular plasticity. This is evident in the fact that several parameters of intestinal metabolism are rapidly upregulated by enteral feeding, typically within 30 min to 2 h; these parameters include blood flow, oxygen consumption, protein synthesis and nutrient transport (24 28). Thus, the question is whether the acute increase in circulating GLP-2 concentration after enteral feeding is linked to the stimulation of acute metabolic actions in the intestine. In a recent study to explore this issue in the TPN-fed piglet model, we measured the rates of intestinal blood flow, protein synthesis, cell proliferation and villus morphology in three groups of enterally or TPN-fed piglets for 24 and 48 h (29). We found that portal blood flow decreases rapidly after starting TPN ( 30% within 6 h), and that protein mass, protein synthesis and villus height in the jejunum are significantly lower after only 24 h of TPN. Interestingly, we also found that inducible nitric oxide synthase activity in the jejunum decreases after 24 h of TPN. Plasma GLP-2 concentrations are significantly lower ( 50%) after 24 h of TPN; unfortunately samples were not collected at earlier time points. These results coupled with previous piglet studies suggest that rapid upregulation in blood flow and protein synthesis are sensitive early metabolic indicators that precede the mucosal structure and composition changes in response to enteral nutrition. Moreover, the temporal changes in these early metabolic indicators and circulating GLP-2 concentration in the postprandial period are consistent with the hypothesis that these two phenomena are functionally linked. What has not been evident from the coincident changes observed in plasma GLP-2 and intestinal metabolism after enteral feeding is the acute nutrient-independent intestinal responsiveness to GLP-2. To investigate this question, we measured the acute changes in intestinal blood flow and substrate metabolism in TPN-fed piglets during a short-term (4-h) intravenous infusion of a pharmacological GLP-2 dose (30). During GLP-2 infusion, we observed a rapid (within 10 min) upregulation of portal blood flow that peaked after 45 min and plateaued at 25% above the saline baseline. We also found that GLP-2 treatment increased portal glucose uptake ( 90%) and protein synthesis ( 125%) during the 4-h infusion. More intriguing, however, was the observation that GLP-2 increased the jejunal constitutive nitric oxide synthase (NOS) activity and protein abundance of endothelial NOS. Subsequent experiments showed that coinfusion with the NOS-inhibitor, L-nitro-arginine methyl ester, completely blocked the GLP-2- stimulation of portal blood flow and glucose uptake suggesting that these effects occur via a nitric-oxide dependent mechanism. These findings indicate that key metabolic functions, which are closely linked to and precede changes in intestinal growth, are rapidly increased in response to increased circulating GLP-2 and this occurs in the absence of luminal nutrients. Yet more importantly, the results implicate nitric oxide as a possible signaling molecule in GLP-2 receptor function and may provide clues as to the precise cellular localization of the receptor, which has been the subject of conflicting reports (31,32). The evidence to date on GLP-2R function suggests that downstream secondary mediators are involved in a heterotypic cellular mechanism of action. This is based on reports of its localization in human enteroendocrine cells and murine enteric neurons, whereas the changes in cellular function in

4 NUTRITION AND NEONATAL INTESTINAL ADAPTATION 3715 and preventing intestinal atrophy in TPN-fed neonates. Furthermore, the results suggest that GLP-2 may play a role in the regulation of blood flow and become therapeutically useful for maintenance of intestinal function in conditions of reduced intestinal perfusion or ischemia. LITERATURE CITED FIGURE 2 Schematic overview of hypothetical model for intestinal mucosal GLP-2 receptor (GLP-2R) signaling. response to GLP-2 have been confined largely to other intestinal cell types (e.g., stimulating crypt cell proliferation and inhibiting enterocyte apoptosis). We speculate that GLP-2 interacts with its receptor on target cells to stimulate endogenous synthesis of NO via constitutive NOS, which in turn upregulates blood flow and substrate utilization necessary for cell growth and proliferation (Fig. 2). CONCLUSIONS In the recent studies in young piglets, there are several observations that support the idea that GLP-2 is indeed an endocrine link between enteral nutrition and intestinal adaptation. First, the GLP-2 secretory response to enteral nutrition is functional as early as late gestation. Second, parallel changes in intestinal growth and circulating GLP-2 occur in response to the quantity and composition of enteral nutrition after birth. Third, the acute temporal changes in intestinal metabolism and circulating GLP-2 concentrations in response to enteral nutrition are also generally coincident. However, in contrast, the lack of intestinal trophic responses to both pharmacological GLP-2 concentrations in the fetus and weanling pigs and to physiological GLP-2 concentrations in neonates raise doubts concerning the physiological relevance of GLP-2 as a enterally mediated trophic signal. A more definitive answer to this question will require further studies testing the intestinal response to enteral nutrition coupled with various experimental approaches to block GLP-2 action, including immunoneutralization of circulating GLP-2, inhibition of GLP-2 secretion, antagonism of GLP-2 receptor function or targeted disruption of the GLP-2 receptor gene. Despite the equivocal evidence for a physiological link between enteral nutrition and intestinal growth, it is apparent that therapeutic doses of GLP-2 are highly efficacious in stimulating growth 1. Kieffer, T. J. & Habener, J. L. (1999) The glucagon-like peptides. Endocr. Rev. 20: Burrin, D. G., Stoll, B. & Guan, X. (2003) Glucagon-like peptide 2 function in domestic animals. Domest. Anim. Endocrinol. 23: Drucker, D. J. (2002) Gut adaptation and the glucagon-like peptides. Gut 50: Drucker, D. J., Ehrlich, P., Asa, S. L. & Brubaker, P. L. (1996) Induction of intestinal epithelial proliferation by glucagon-like peptide 2. 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