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Zinc deficiency and diarrhea: Is uroguanylin the culprit?
January 1998
SELECTED SUMMARIES
is usually the equivalent of distant disease, with a mean life expectancy of 7 months and of ,3 years even if the recurrence is resectable (Cancer 1974;34:1278–1292, Br J Surg 1994;81:7–19). Pelvic or anastomotic recurrence after resection of a rectal carcinoma carries a slightly more favorable prognosis, but is locally resectable 20% of the time (Br J Surg 1994;81:7–19). In the patients undergoing resection, even when the recurrence is asymptomatic and has been identified through carcinoembryonic antigen surveillance, resectability is about 30%–50% and long-term survival about 25% (Br J Surg 1996;83:293– 304). Third, for patients with metastatic spread confined to the liver, about one third meet criteria for consideration for resection, about half of these are resectable, and long-term survival in this highly selected group is about 25% (Dis Colon Rectum 1988;31:1–4, J Clin Oncol 1991;9:1105–1112). In this group of highly selected patients who meet criteria for resection, optimistic reports have suggested that 25%–35% may be cured (Dis Colon Rectum 1988;31:1–4, J Clin Oncol 1991;9:1105–1112). For patients with isolated metastases to the lung, the outlook is more dismal (Curr Probl Cancer 1987;11:259– 356). All of these considerations are consistent with the results of the present study. The observations reported in the current study provide a hard glimpse of the total picture of potentially treatable recurrences: with a relatively intensive program of follow-up, maintained for an average of .5 years (the period in which virtually all recurrences present), local or distant recurrence amenable to resection for cure could be identified in ,20% of patients with any recurrence. About 12% of patients with recurrence had such surgery performed when the recurrence was asymptomatic. The benefit of earlier detection by more intensive surveillance was identifiable for, at most, 8% of patients who would have a recurrence and for only 2.0% of all patients undergoing surveillance. For those already pessimistic about our ability to treat and cure recurrences, these considerations would suggest that it is not ‘‘medically indicated’’ to perform intensive postoperative surveillance. For those who focus interest on the costs of care, the small and unproved benefit of intensive programs of follow-up would almost certainly lead to the conclusion that monies used for them (eight visits per patient per 5 years using specialist or primary physician time and laboratory and imaging facilities) are being spent for no good reason. The authors stopped short of embracing this conclusion, and I agree with their reticence to do so. Unless we follow up our patients closely, we will not be able to evaluate the contributions of more sensitive tests for identifying small persistent deposits of tumor or recurrences at the earliest, asymptomatic stages of detectability. Further, most patients will almost certainly feel a sense of abandonment should they be told that minimal follow-up programs and evaluations are necessary. In this regard, it would have been very interesting if the authors had information regarding the psychological and emotional experience of the different programs of surveillance. Their data certainly support the case that, for now, patients with asymptomatic recurrences do not fare better than those with symptomatic recurrences. In addition, their data suggest that no harm will necessarily occur by individualizing follow-up programs for individual practices. DAVID I. SOYBEL, M.D.
ZINC DEFICIENCY AND DIARRHEA: IS UROGUANYLIN THE CULPRIT? Blanchard RK, Cousins RJ (Department of Food Science and Human Nutrition, University of Florida Center for Nutritional Sciences, Gainesville, Florida). Upregulation of rat intestinal
221
uroguanylin mRNA by dietary zinc restriction. Am J Physiol 1997;272:G972–G978. Despite the well-documented association between dietary zinc deficiency and diarrhea, the precise mechanism responsible for this association has been elusive. This intriguing new work by Blanchard and Cousins may help to close that gap in our knowledge by its demonstration that zinc deficiency leads to increased expression of uroguanylin, also known as guanylate cyclase–activating peptide II. Previous studies by Cousins et al. using differential display polymerase chain reaction (PCR) in rats showed the rat homologue of human uroguanylin as one of several complementary DNAs (cDNAs) that were increased as a result of a zinc-deficient diet (Proc Natl Acad Sci USA 1996;93:6863–6868). In the present study, the authors directly tested the hypothesis that zinc deficiency leads to overexpression of uroguanylin gene expression. They began with the previously identified clone that was overexpressed in zinc deficiency and shared a high degree of homology with uroguanylin cDNA sequences of human and opossum. PCR primers were designed from this sequence and the 58 portion of the rat cDNA was cloned using a 58 RACE procedure. Rats were provided free access to a diet that was either zinc adequate (30 mg zinc/kg) or zinc deficient (,1 mg zinc/kg), and a third group was pair-fed a zinc-adequate diet in amounts equal to food consumption in the zinc-deficient group, to control for the anorexia associated with zinc deficiency. After 15 days, the rats were killed, small intestinal mucosa was harvested for quantitative Northern analysis, and sections of duodenum, jejunum, ileum, and ascending colon were taken for analysis of regional distribution of uroguanylin message. A 651-base cDNA sequence cloned from the original one was up-regulated during zinc deficiency. The predicted amino acid sequence from the open reading frame for the rat sequence predicts a 106–amino acid protein, and the C-terminal 15 residues, which correspond to bioactive uroguanylin, showed a high degree of homology (93%–100%) with the sequence from other species. Using a probe constructed from the original clone, the investigators showed that zinc deficiency resulted in a 2.5-fold increase in the abundance of uroguanylin messenger RNA (mRNA) in the small intestine, whereas rats on the normal diet or those in the pair-fed group showed no change in uroguanylin mRNA levels. On Northern blots, uroguanylin mRNA in normal rats was most abundant in the jejunum, followed by duodenum and ileum. The next strongest signals were seen in colon and thymus, with only faint bands seen for stomach, kidney, testis, and spleen. Other tissues studied showed insignificant signals. Zinc deficiency did not alter uroguanylin mRNA levels in kidney or thymus. Comment. Diarrhea is one of several symptoms of zinc deficiency, along with weight loss, skin lesions, hair loss, and impaired immune function. Speculations on the mechanisms responsible for diarrhea in zinc-deficient patients derived primarily from the observations that zinc deficiency disrupts small intestinal architecture and reduces brush border hydrolase activity and the resolution of these abnormalities on zinc supplementation (through either the oral or parenteral
222
SELECTED SUMMARIES
route). This had led to the idea that zinc deficiency results in diarrhea caused by small intestinal malabsorption. Furthermore, zinc’s contribution to membrane structure suggested that deficiency of this trace element may influence intestinal transport by altering such functions as the activity of apical ion channels and mucosal permeability to ions ( J Pediatr Gastroenterol Nutr 1984;3:608–612, Gut 1996;39:416– 422). The work of Blanchard and Cousins adds a new dimension to the zinc/diarrhea story by providing a link, at the level of gene expression, between zinc deficiency and a molecule that is being increasingly recognized as a potential regulator of intestinal water and electrolyte transport. Uroguanylin was first described as a peptide closely related to guanylin, the endogenous ligand of the apical receptor for Escherichia coli heat-stable enterotoxin, which is also the membranebound guanylate cyclase C that increases intracellular levels of cyclic guanosine monophosphate (cGMP). Uroguanylin’s name derives from its initial isolation from opossum and human urine; like its sibling guanylin, it stimulates cGMP levels in T-84 cells and elevates short-circuit current, a measure of active Cl2 secretion (Proc Natl Acad Sci USA 1993; 90:10464–10468, Am J Physiol 1994;266:F342– F348). The implication of these new findings is, therefore, that diarrhea in zinc deficiency may result from increased cGMPstimulated Cl2 secretion in intestinal epithelial cells, a process that is the major driving force for active fluid secretion into the intestinal lumen. As the investigators rightly note, this conclusion must be verified with experiments that measure the effect of zinc deficiency on the circulating levels of uroguanylin, and ultimately on cGMP production in enterocytes, to determine if zinc deficiency does indeed affect the physiological pathways that influence intestinal fluid and electrolyte transport. There is, however, other indirect support for zinc’s role in intestinal epithelial ion transport. Rodriguez et al. (Gut 1996;39:416–422) recently showed that malnourished guinea pigs whose diets are supplemented by pharmacological doses of zinc do not exhibit the elevated basal short-circuit current (and increased tissue permeability) that is typical of this condition. Based on the study of Blanchard and Cousins, one mechanism to account for this effect could be suppression of intestinal uroguanylin levels after zinc supplementation that reduces basal ion secretion in the malnourished animals. Future studies that will likely be initiated based on these new results should be helpful in better understanding not only the pathogenesis of diarrhea induced by zinc deficiency but also the general mechanisms involved in nutrient effects on gene expression in the gastrointestinal tract. Thus, it will be very interesting to know whether zinc deficiency also alters gene expression of the closely related peptide, guanylin; if not, this may shed light on how this particular signal exerts differential effects on transcriptional regulation of closely related genes. The question of how the signal is transmitted from dietary zinc to the cells that produce uroguanylin will also be of great interest—is it systemic, or at the local level of the intestine? A much larger question that may be answered at some point is the adaptive significance, if any, of the association between dietary zinc status and uroguanylin-regulated fluid and electrolyte transport. The eventual therapeutic application of all of this basic knowledge may come in the development of new approaches in the treatment of diarrheal disorders that result from defective activity of cellular mediators that lead to intestinal secretion: can zinc regulation of uroguanylin gene expression be used to either minimize or promote intestinal fluid transport? The demonstration that uroguanylin is expressed at high levels in the rat small intestine is consistent with other recent reports (FEBS Lett 1996;398:170–174, Regul Pept 1997;68:45–56, Am J Physiol 1997;273:G93–G105) and with levels
GASTROENTEROLOGY Vol. 114, No. 1
reported in opossum (Biochem Biophys Res Commun 1996;219:457– 462). Interestingly, in both species, uroguanylin message seems to be greater in the intestine than the kidney, despite the initial isolation of the peptide from urine. This suggests that uroguanylin is released from intestinal tissues, including small and large bowel, with subsequent filtration in the kidney, perhaps to coordinate an intestinalrenal response to changes in systemic fluid and electrolyte balance (Am J Physiol 1996;270:G708–G716). The future for understanding the significance of this very interesting peptide in normal and pathological conditions in the gut is getting brighter all the time. HANNAH V. CAREY, Ph.D.
Reply. We appreciate the comments on our papers describing the up-regulation of uroguanylin expression in zinc deficiency. As indicated in the summary, this observation was made while carrying out differential mRNA screening to identify genes that are regulated by differences in dietary zinc status. It should be pointed out that an association between zinc deficiency and diarrhea in humans has been known for nearly 20 years (in Hurley LS, ed. Trace elements in man and animals. 4th ed. New York: Plenum Press, 1981;73–76; Acta Paediatr Suppl 1992;381:82–86). Recently, convincing evidence has shown that zinc supplementation of children markedly reduced the incidence of diarrhea in studies performed in India and Mexico (Am J Clin Nutr 1997;65:13–19, New Engl J Med 1995;333:839–844). We speculate that the higher incidence of diarrhea and its reduction by zinc supplementation is the result of a change in the production of uroguanylin. It should be noted that zinc-deficient rats, the model used in our experiments, do not usually experience diarrhea. This is likely due to the large rat cecum, which can serve as a reservoir for fluid reabsorption under conditions that would otherwise produce secretory diarrhea ( J Pharmacol Methods 1990;24:59–71). Nonetheless, when there is a costimulus, e.g., an interleukin 1 injection, diarrhea can be induced in zinc-deficient rats but not in zinc-adequate rats ( J Nutr 1997;127:1729–1736). A stimulus of this type may further elevate uroguanylin production, but this association remains to be tested. A mechanism for the up-regulation of uroguanylin expression in zinc deficiency is unclear at the present time. Zinc can regulate genes through an association with a metal response element transcription factor, which acts to alter transcription rates of genes controlled by metal response elements in their promoters. This phenomenon is usually positive, i.e., extra zinc increases transcription, but it is also possible that some genes are activated when the amount of zinc present is less than optimal. An alternate hypothesis is that because uroguanylin is a peptide hormone that requires processing, activities of zinc-requiring membrane metallopeptidases (FASEB J 1997;11:355– 364) are decreased in zinc deficiency. The increase in uroguanylin mRNA expression could then be a response to compensate for a lack of processing of the prouroguanylin to the active peptide. RAYMOND K. BLANCHARD ROBERT J. COUSINS
Editor’s Comment: The recent report (N Engl J Med 1997;333: 839–844) that zinc supplementation for infants and young children with acute diarrhea was associated with clinically important reduction in the duration and severity of diarrhea is consistent with Dr. Carey’s comments regarding the possible clinical relevance of observed up-regulation of uroguanylin in zinc deficiency.
SELECTED SUMMARIES
is usually the equivalent of distant disease, with a mean life expectancy of 7 months and of ,3 years even if the recurrence is resectable (Cancer 1974;34:1278–1292, Br J Surg 1994;81:7–19). Pelvic or anastomotic recurrence after resection of a rectal carcinoma carries a slightly more favorable prognosis, but is locally resectable 20% of the time (Br J Surg 1994;81:7–19). In the patients undergoing resection, even when the recurrence is asymptomatic and has been identified through carcinoembryonic antigen surveillance, resectability is about 30%–50% and long-term survival about 25% (Br J Surg 1996;83:293– 304). Third, for patients with metastatic spread confined to the liver, about one third meet criteria for consideration for resection, about half of these are resectable, and long-term survival in this highly selected group is about 25% (Dis Colon Rectum 1988;31:1–4, J Clin Oncol 1991;9:1105–1112). In this group of highly selected patients who meet criteria for resection, optimistic reports have suggested that 25%–35% may be cured (Dis Colon Rectum 1988;31:1–4, J Clin Oncol 1991;9:1105–1112). For patients with isolated metastases to the lung, the outlook is more dismal (Curr Probl Cancer 1987;11:259– 356). All of these considerations are consistent with the results of the present study. The observations reported in the current study provide a hard glimpse of the total picture of potentially treatable recurrences: with a relatively intensive program of follow-up, maintained for an average of .5 years (the period in which virtually all recurrences present), local or distant recurrence amenable to resection for cure could be identified in ,20% of patients with any recurrence. About 12% of patients with recurrence had such surgery performed when the recurrence was asymptomatic. The benefit of earlier detection by more intensive surveillance was identifiable for, at most, 8% of patients who would have a recurrence and for only 2.0% of all patients undergoing surveillance. For those already pessimistic about our ability to treat and cure recurrences, these considerations would suggest that it is not ‘‘medically indicated’’ to perform intensive postoperative surveillance. For those who focus interest on the costs of care, the small and unproved benefit of intensive programs of follow-up would almost certainly lead to the conclusion that monies used for them (eight visits per patient per 5 years using specialist or primary physician time and laboratory and imaging facilities) are being spent for no good reason. The authors stopped short of embracing this conclusion, and I agree with their reticence to do so. Unless we follow up our patients closely, we will not be able to evaluate the contributions of more sensitive tests for identifying small persistent deposits of tumor or recurrences at the earliest, asymptomatic stages of detectability. Further, most patients will almost certainly feel a sense of abandonment should they be told that minimal follow-up programs and evaluations are necessary. In this regard, it would have been very interesting if the authors had information regarding the psychological and emotional experience of the different programs of surveillance. Their data certainly support the case that, for now, patients with asymptomatic recurrences do not fare better than those with symptomatic recurrences. In addition, their data suggest that no harm will necessarily occur by individualizing follow-up programs for individual practices. DAVID I. SOYBEL, M.D.
ZINC DEFICIENCY AND DIARRHEA: IS UROGUANYLIN THE CULPRIT? Blanchard RK, Cousins RJ (Department of Food Science and Human Nutrition, University of Florida Center for Nutritional Sciences, Gainesville, Florida). Upregulation of rat intestinal
221
uroguanylin mRNA by dietary zinc restriction. Am J Physiol 1997;272:G972–G978. Despite the well-documented association between dietary zinc deficiency and diarrhea, the precise mechanism responsible for this association has been elusive. This intriguing new work by Blanchard and Cousins may help to close that gap in our knowledge by its demonstration that zinc deficiency leads to increased expression of uroguanylin, also known as guanylate cyclase–activating peptide II. Previous studies by Cousins et al. using differential display polymerase chain reaction (PCR) in rats showed the rat homologue of human uroguanylin as one of several complementary DNAs (cDNAs) that were increased as a result of a zinc-deficient diet (Proc Natl Acad Sci USA 1996;93:6863–6868). In the present study, the authors directly tested the hypothesis that zinc deficiency leads to overexpression of uroguanylin gene expression. They began with the previously identified clone that was overexpressed in zinc deficiency and shared a high degree of homology with uroguanylin cDNA sequences of human and opossum. PCR primers were designed from this sequence and the 58 portion of the rat cDNA was cloned using a 58 RACE procedure. Rats were provided free access to a diet that was either zinc adequate (30 mg zinc/kg) or zinc deficient (,1 mg zinc/kg), and a third group was pair-fed a zinc-adequate diet in amounts equal to food consumption in the zinc-deficient group, to control for the anorexia associated with zinc deficiency. After 15 days, the rats were killed, small intestinal mucosa was harvested for quantitative Northern analysis, and sections of duodenum, jejunum, ileum, and ascending colon were taken for analysis of regional distribution of uroguanylin message. A 651-base cDNA sequence cloned from the original one was up-regulated during zinc deficiency. The predicted amino acid sequence from the open reading frame for the rat sequence predicts a 106–amino acid protein, and the C-terminal 15 residues, which correspond to bioactive uroguanylin, showed a high degree of homology (93%–100%) with the sequence from other species. Using a probe constructed from the original clone, the investigators showed that zinc deficiency resulted in a 2.5-fold increase in the abundance of uroguanylin messenger RNA (mRNA) in the small intestine, whereas rats on the normal diet or those in the pair-fed group showed no change in uroguanylin mRNA levels. On Northern blots, uroguanylin mRNA in normal rats was most abundant in the jejunum, followed by duodenum and ileum. The next strongest signals were seen in colon and thymus, with only faint bands seen for stomach, kidney, testis, and spleen. Other tissues studied showed insignificant signals. Zinc deficiency did not alter uroguanylin mRNA levels in kidney or thymus. Comment. Diarrhea is one of several symptoms of zinc deficiency, along with weight loss, skin lesions, hair loss, and impaired immune function. Speculations on the mechanisms responsible for diarrhea in zinc-deficient patients derived primarily from the observations that zinc deficiency disrupts small intestinal architecture and reduces brush border hydrolase activity and the resolution of these abnormalities on zinc supplementation (through either the oral or parenteral
222
SELECTED SUMMARIES
route). This had led to the idea that zinc deficiency results in diarrhea caused by small intestinal malabsorption. Furthermore, zinc’s contribution to membrane structure suggested that deficiency of this trace element may influence intestinal transport by altering such functions as the activity of apical ion channels and mucosal permeability to ions ( J Pediatr Gastroenterol Nutr 1984;3:608–612, Gut 1996;39:416– 422). The work of Blanchard and Cousins adds a new dimension to the zinc/diarrhea story by providing a link, at the level of gene expression, between zinc deficiency and a molecule that is being increasingly recognized as a potential regulator of intestinal water and electrolyte transport. Uroguanylin was first described as a peptide closely related to guanylin, the endogenous ligand of the apical receptor for Escherichia coli heat-stable enterotoxin, which is also the membranebound guanylate cyclase C that increases intracellular levels of cyclic guanosine monophosphate (cGMP). Uroguanylin’s name derives from its initial isolation from opossum and human urine; like its sibling guanylin, it stimulates cGMP levels in T-84 cells and elevates short-circuit current, a measure of active Cl2 secretion (Proc Natl Acad Sci USA 1993; 90:10464–10468, Am J Physiol 1994;266:F342– F348). The implication of these new findings is, therefore, that diarrhea in zinc deficiency may result from increased cGMPstimulated Cl2 secretion in intestinal epithelial cells, a process that is the major driving force for active fluid secretion into the intestinal lumen. As the investigators rightly note, this conclusion must be verified with experiments that measure the effect of zinc deficiency on the circulating levels of uroguanylin, and ultimately on cGMP production in enterocytes, to determine if zinc deficiency does indeed affect the physiological pathways that influence intestinal fluid and electrolyte transport. There is, however, other indirect support for zinc’s role in intestinal epithelial ion transport. Rodriguez et al. (Gut 1996;39:416–422) recently showed that malnourished guinea pigs whose diets are supplemented by pharmacological doses of zinc do not exhibit the elevated basal short-circuit current (and increased tissue permeability) that is typical of this condition. Based on the study of Blanchard and Cousins, one mechanism to account for this effect could be suppression of intestinal uroguanylin levels after zinc supplementation that reduces basal ion secretion in the malnourished animals. Future studies that will likely be initiated based on these new results should be helpful in better understanding not only the pathogenesis of diarrhea induced by zinc deficiency but also the general mechanisms involved in nutrient effects on gene expression in the gastrointestinal tract. Thus, it will be very interesting to know whether zinc deficiency also alters gene expression of the closely related peptide, guanylin; if not, this may shed light on how this particular signal exerts differential effects on transcriptional regulation of closely related genes. The question of how the signal is transmitted from dietary zinc to the cells that produce uroguanylin will also be of great interest—is it systemic, or at the local level of the intestine? A much larger question that may be answered at some point is the adaptive significance, if any, of the association between dietary zinc status and uroguanylin-regulated fluid and electrolyte transport. The eventual therapeutic application of all of this basic knowledge may come in the development of new approaches in the treatment of diarrheal disorders that result from defective activity of cellular mediators that lead to intestinal secretion: can zinc regulation of uroguanylin gene expression be used to either minimize or promote intestinal fluid transport? The demonstration that uroguanylin is expressed at high levels in the rat small intestine is consistent with other recent reports (FEBS Lett 1996;398:170–174, Regul Pept 1997;68:45–56, Am J Physiol 1997;273:G93–G105) and with levels
GASTROENTEROLOGY Vol. 114, No. 1
reported in opossum (Biochem Biophys Res Commun 1996;219:457– 462). Interestingly, in both species, uroguanylin message seems to be greater in the intestine than the kidney, despite the initial isolation of the peptide from urine. This suggests that uroguanylin is released from intestinal tissues, including small and large bowel, with subsequent filtration in the kidney, perhaps to coordinate an intestinalrenal response to changes in systemic fluid and electrolyte balance (Am J Physiol 1996;270:G708–G716). The future for understanding the significance of this very interesting peptide in normal and pathological conditions in the gut is getting brighter all the time. HANNAH V. CAREY, Ph.D.
Reply. We appreciate the comments on our papers describing the up-regulation of uroguanylin expression in zinc deficiency. As indicated in the summary, this observation was made while carrying out differential mRNA screening to identify genes that are regulated by differences in dietary zinc status. It should be pointed out that an association between zinc deficiency and diarrhea in humans has been known for nearly 20 years (in Hurley LS, ed. Trace elements in man and animals. 4th ed. New York: Plenum Press, 1981;73–76; Acta Paediatr Suppl 1992;381:82–86). Recently, convincing evidence has shown that zinc supplementation of children markedly reduced the incidence of diarrhea in studies performed in India and Mexico (Am J Clin Nutr 1997;65:13–19, New Engl J Med 1995;333:839–844). We speculate that the higher incidence of diarrhea and its reduction by zinc supplementation is the result of a change in the production of uroguanylin. It should be noted that zinc-deficient rats, the model used in our experiments, do not usually experience diarrhea. This is likely due to the large rat cecum, which can serve as a reservoir for fluid reabsorption under conditions that would otherwise produce secretory diarrhea ( J Pharmacol Methods 1990;24:59–71). Nonetheless, when there is a costimulus, e.g., an interleukin 1 injection, diarrhea can be induced in zinc-deficient rats but not in zinc-adequate rats ( J Nutr 1997;127:1729–1736). A stimulus of this type may further elevate uroguanylin production, but this association remains to be tested. A mechanism for the up-regulation of uroguanylin expression in zinc deficiency is unclear at the present time. Zinc can regulate genes through an association with a metal response element transcription factor, which acts to alter transcription rates of genes controlled by metal response elements in their promoters. This phenomenon is usually positive, i.e., extra zinc increases transcription, but it is also possible that some genes are activated when the amount of zinc present is less than optimal. An alternate hypothesis is that because uroguanylin is a peptide hormone that requires processing, activities of zinc-requiring membrane metallopeptidases (FASEB J 1997;11:355– 364) are decreased in zinc deficiency. The increase in uroguanylin mRNA expression could then be a response to compensate for a lack of processing of the prouroguanylin to the active peptide. RAYMOND K. BLANCHARD ROBERT J. COUSINS
Editor’s Comment: The recent report (N Engl J Med 1997;333: 839–844) that zinc supplementation for infants and young children with acute diarrhea was associated with clinically important reduction in the duration and severity of diarrhea is consistent with Dr. Carey’s comments regarding the possible clinical relevance of observed up-regulation of uroguanylin in zinc deficiency.