Alterations in serum and antral gastrin levels in genetically diabetic mice

GASTROENTEROLOGY

77:1276-1282,1979

Alterations in Serum and Antral Gastrin Levels in Genetically Diabetic Mice LENARD M. LICHTENBERGER RAMASWAMY

and

KRISHNAMURTHY

Departments of Physiology and Medicine, The University Houston, Texas

Many gastrointestinal structural and functional properties are known to be altered in diabetes. In this study, we investigated whether serum and tissue gastrin levels are abnormally altered in a strain of genetically diabetic mice (C57BL/KS]). Both serum and antral gastrin concentration were found to be significantly increased 3.4- and n-fold above normal values in diabetic mice fed ad Jibitum. The increase in tissue gastrin concentration is most probably due to an increase in both cellular gastrin content and G-cell number, since the latter property is increased 130% in diabetic animals. Pair feeding studies demonstrated that diabetes associated hyperphagia is not a major factor in inducing these endocrine changes, since antral and serum gastrin are still significantly elevated above normal in diabetic animals fed a restricted diet. G-cell number, however, is not significantly increased above normal values in pair fed diabetic mice. The peak serum gastrin concentration after a meal and the duration of postprandial hypergastrinemia are also significantly increased above normal in diabetic animals. Gel filtration chromatography studies indicate that the antral mucosoe of normal and diabetic mice have identical molecular forms of the hormone. It is therefore concluded that an&al and serum gastrin concentration are increased in genetically diabetic mice due to both dietary alterations and other, as yet undefined, factors specific for the disease, and that the result-

Received January 29,1979. Accepted June Z&1979. Address requests for reprints to: Lenard M. Lichtenberger, M.D., Department of Physiology, The University of Texas Medical School at Houston, P.O. Box 20706, Houston, Texas 77030. This work was supported in part by NIH Grant AM-19876 and AM-20686. The authors would like to thank Miss Leslie Koziar, Mrs. Robin Bailey, and Mrs. Elizabeth Guffy for their technological assistance. 0 1979 by the American Gastroenterological Association OOlS-5085/79/121276-07$02.00

of Texas Medical School at Houston,

ant hypergastrinemia may contribute to some of the gastrointestinal alterations seen in diabetes.

Diabetes has been reported to be associated with abnormalities in both gastrointestinal structure and function. It has been documented that disorders in gastrointestinal motility are a common problem among diabetic patients with gastric retention occurring in ZO-35% and diarrhea being reported to occur in 6% of the diabetic population.‘-e Pancreatic and gastric secretory capacity are also impaired in diabetes. Pancreatic exocrine insufficiency has been reported to occur in 40-100% of the diabetic patients tested, with the major abnormality being found in abnormally low pancreatic enzyme output.7-8 Similarly, it has been reported that diabetic patients have a below normal capacity to secrete gastric acid under both basal and stimulated conditions.9-” Several studies have indicated that between 17 and 70% of the diabetic patients tested secrete no acid in response to a histamine challenge.‘-I1 This diabetes-associated decrease in gastric secretory capacity has, in part, been attributed to the finding that a high percentage of patients have some form of atrophic gastritis.9”2 Gastrointestinal abnormalities in diabetes have been studied to only a limited degree in animal models of the disease. Several laboratories have reported that the active transport of glucose is enhanced in drug-induced diabetes, 13.*4but not in genetically diabetic anima1s.l’ Brush-border disaccharidase activity appears to be increased in all the animal models tested to date.1a,‘7 One finding of particular interest is that it has been reported that the intestinal epithelium is hyperproliferative in drug-induced diabetic animals.‘B-zo Pair-feeding studies have indicated that the diabetes-induced increase in mucosal weight, surface area, and cell proliferation cannot be solely attributable to the enhancement of food intake

December 1979

which is known to be associated with the disease.” This suggests that other, possibly humoral, factors may be responsible for stimulating mucosal growth in diabetes. The pathophysiologic mechanism(s) responsible for mediating these changes in diabetes in humans and the laboratory animals are unknown. It is certainly well established that many of these gastrointestinal properties (i.e., gastric and pancreatic secretion, gastric emptying, glucose transport, disaccharidase activity, and gastrointestinal growth)21.ZZare under the influence of the antral hormone, gastrin. In this study, therefore, we will focus our investigations on possible alterations in tissue and serum gastrin concentration in ad libitum and pair fed genetically diabetic animals, in order to determine whether alterations in gastrin levels may mediate the associated gastrointestinal abnormalities. The effect of different feeding schedules was examined to be able to discriminate between specific diabetes induced alterations in gastrin levels from those induced by hyperphagia, which is associated with the disease. The animal model selected for study is the obese, genetically diabetic mouse (C57 BL/KSJ) which carries the recessive diabetic gene, db. This particular strain is considered to be similar in many respects to “maturity onset” diabetes in humans, since the homozygous db/db diabetic mice are obese, hyperglycemic, hyperinsulinemic, hyperglucagonemic, and insulin resistant.z3.24

Methods Breeding pairs of normal and diabetic mice of the C57 BL/KSJ strain were originally purchased from Jackson Laboratory (Bar Harbor, Maine). From this breeding stock, a colony of genetically diabetic and normal mice was raised in our animal facility. The close linkage of the recessive genes for color coat, m, and diabetes, db, allowed easy identification of the homozygous (+m/+m) and heterozygous (+m/db+) normals which are lean and brown (misty) colored, and lean and black colored, respectively. Diabetes (db+/db+), on the other hand, are obese mice with a black color coat.Z3.*4Diabetic mice and their normal (homozygous and heterozygous) littermates were then studied at 2-3 mo of age. Whenever possible, male and female animals were divided equally among the study group. The experiments to be described below were subdivided into the three studies: Study I. Normal and diabetic mice were fed ad libitum on chow up till the time of sacrifice, after which serum and antral gastrin concentrations were determined by radioimmunoassay, and the number of immunoreactive G-cells was quantitated by immunocytochemistry.

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Study II. Normal and diabetic mice were pair fed a diet of 4 g of chow per day (the average daily consumption of normal mice) over an 18-day period, after which serum and tissue gastrin concentration, as well as G-cell number, were determined. Study III. Normal and diabetic mice were fasted for 18 hr. At this time, four to five mice from each group were sacrificed, and the remaining animals were allowed to feed ad libitum on chow for a l-hr period (after which the food was removed), and sacrificed at 1,2,and 3 hr after the onset of feeding. The serum gastrin concentration was then determined. The following techniques were employed in these studies.

Gas&in

Extraction

and Radioimmunoassay

All animals were sacrificed by decapitation, at which time blood was collected. The stomach was then removed, slit open along the greater curvature, rinsed in saline, and pinned out mucosal side up on a sheet of dental wax. A piece of distal antrum, along the lesser curvature and bordering on the pyloric junction, was then dissected free, weighed, and homogenized in 2 ml of boiling water. The homogenate was then incubated at 96°C for 20 min, centrifuged at 500 g, and the supernatant was collected. The antral extract and serum samples were then stored at -20°C until gastrin radioimmunoanalysis. Gastrin radioimmunoassay was performed by a modification of the technique of Yalow and Berson, as described by Walsh.Z5-28 Antisera No. 1296 (donated by Dr. John H. Walsh of CURE) was used as both standard and label. Gastrin was iodinated by the chloramine T technique and purified as described by Stadil and Rehfeld.29

G-Cell Immunocytochemistry A small strip (2 x 4 mm) of distal gastric antrum was mounted, mucosal side up, on filter paper and immediately placed in a modified Bouins fixative (containing only 1% glacial acetic acid). The tissue was then embedded in pariffin and sectioned (6 pm) using standard morphologic techniques. The sections were then deparaffinized and hydrated and placed in humidified incubation chambers designed for immunohistochemistry. The G-cells were then specifically stained, using an indirect immunoperoxidase method, which was a modification of a gastrin immunohistochemical technique described previously.” Briefly, the method entails layering gastrin-specific antisera (1:40 dilution of antisera No. 4, raised in rabbits in our laboratory against 15-leu G-17 gastrin) over the sections for a 30-min incubation period. At this time, the slides were washed for 1 hr in 0.01 M phosphate-buffered saline (pH 7.2), followed by the addition of peroxidase labeled goat antirabbit gamma-globulin (1:20) for a 30-min incubation period. This step was followed by another I-hr washing period in buffer, and the reaction was developed by incubating the sections in 3’3’-diaminobenzidine (30 mg%) in 0.05 M Tris-saline (pH 7.6) containing H,O, (0.001%) for a 30-min period.“” The sections were washed

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LJCHTENBERGER

in PBS for 15 min, followed by a second 18min wash in Tris-water, and then osmicated for 5 min in 2% 0~0, in Mollinig buffer. The sections were then dehydrated with graded alcohols, cleaned, and coverslipped with Permount. The cells whose cytoplasm had been stained with a brown pigment were considered to be G-cells, and their number along a given length of antral muscosae (as determined with a micrometer eyepiece) was quantitated in double-blind fashion by two independent observers. The specificity of the technique was controlled for by performing the procedure described above on sections which were initially incubated in either (a) normal rabbit serum (1:40) or (b) gastrin-specific antisera (1:40) that was preincubated with 100 kg/ml of gastrin for 48 hr before the experiment. In both cases no positively stained cells were seen.

Sephadex

GASTROENTEROLOGY

AND RAMASWAMY

mice placed on two different feeding schedules. In the first study normal and diabetic mice were allowed to feed and ad libitum for several weeks, afterwhich the animals were sacrificed and these properties were measured. Food intake measurements indicated that normal mice consumed 3.94 + 0.20 g of chow/day whereas diabetic mice daily consumed approximately twice that amount (7.43 + 0.22 g/day). In order to control for this difference in food consumption and its influence on the endocrine properties being studied, a second study was performed in which all mice had access to only 4 g chow/day (average food intake of controls) for an l&day period, after which the animals were sacrificed and the biochemical and endocrinologic measurements were performed.

Chromatography

Antral extracts (0.2 ml) from normal and diabetic mice (3 animals/group) were applied to a G-50 Superfine Sephadex column (1 X 100 cm) which was being eluted with a 0.02 M Verona1 buffer (pH 8.4) containing 0.02% sodium azide, at a flow rate of 15 ml/hr (1 ml/tube). Plasmatein (0.1 ml), a few grains of dextran blue and 1000 cpm of “‘1 G-171 gastrin were added to the sample before application. The,elution position of antral gastrin was determined by radioimmunoassay. The beginning and the end of the column was indicated by the appearance of void volume marker (dextran blue) and the salt peak (as determined by conductivity measurements) respectively. The position of the Y gastrin reference standard was determined by gamma-counting. The entire chromatography run was performed at 4°C.

Blood

Food Intake The daily food consumption of normal and diabetic mice was measured by placing preweighed food cups containing pulverized chow into hanging basket cages (1 mouse/cage). At the end of a 24-hr period, the food cups, along with the spillage, were weighed, and the difference between the two measurement represented the food intake/day. In addition, the food intake during a I-hr test meal was measured using similar techniques are described above.

Glucose

Blood samples were immediately deproteinized upon collection, and glucose levels were determined by a modified glucose oxidase technique.31

Results In the results to be discussed below we will present data obtained on blood glucose concentration, antral and serum gastrin concentration, and Gcell immunocytochemistry of normal and diabetic

Glucose

Concentration

The blood glucose concentrations of ad libiturn and pair fed normals and diabetic mice are shown in Table 1. It can be seen that although diabetic mice on both dietary schedules were markedly hyperglycemic, the difference in blood sugar levels between normal and obese mice was greater in the ad libitum fed animals. The reasons for the difference in blood glucose concentration between the control groups of the two studies is unclear but may be attributable to the different periods of day the samples were collected.

AntraJ

Blood

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and

Serum

Gastrin

Concentration

The serum and antral gastrin concentration of ad libitum feeding normal and diabetic animals are in either serum or shown in Figure 1A. No difference antral gastrin concentration was detected between normal homozygotes and heterozygotes and therefore, hormone levels of these two groups were pooled in all studies. Serum gastrin concentration is significantly higher in diabetic animals, being 3.4fold greater than hormone levels of normal mice. Similarly, antral gastrin concentration is significantly increased in diabetes being twofold higher in diabetic than normal mice. Placing diabetic mice on a restricted diet did not abolish the differences in gastrin levels between groups. Figure 1B demonstrates that serum gastrin

Table 1.

Blood Glucose Concentration of Ad libitum and Pair Fed Normal and Diabetic Mice

Group

7

Normal Diabetic

9 9

All data expressed

mean

Ad libitum

fed

129 + 5 540 -c 24 mg% glucose

k SEM.

Pair fed 188 -c 12 434 -c 44

December

1979

A. Ad Libitum

GASTRIN

Fed

Gastrin Secretory

Fed *

Figure

IN DIABETES

1279

but significant, 30% increase in G-cell number in freely feeding diabetic animals, in comparison to normal values. In contrast to mice feeding ad libiturn, there is no difference in G-cell number between groups when mice are placed on a restricted diet (Table 1).

r

,w_ 8.Pti

LEVELS

1. Serum and antral gastrin concentration in A. ad libitum fed and b. Pair fed (4 g chow/day for an l&day period) normal and genetically diabetic mice. In this and subsequent figures, the asterisk represents a statistically significant difference at P < 0.05 between groups, and all data are expressed as mean Y&SEM.

concentration is significantly higher in diabetic animals, being 3.6-fold greater than the levels of pair fed controls. The antral gastrin concentration also proved to be significantly greater in diabetic than normal mice in the pair feeding study. The magnitude of difference in tissue hormone concentration between groups, however, was considerably less in animals placed on the restricted diet than measured in ad libitum fed mice. It should be noted that the antral gastrin concentration of the control animals differed between the two studies. It, however, is difficult to say whether this was directly attributable to variations in the feeding schedules or to other aspects of the experiment (i.e., different litters of mice were used in the two studies, animals in the two studies were not sacrificed at precisely the same time of day, etc.).

Response

to a Meal

In this experiment, fasted normal and diabetic mice were allowed to feed on chow for a l-hr period and sacrificed at various periods afterwards for serum gastrin determination. It was subsequently determined that normals and diabetics consumed a comparable quantity of chow (0.73 -C0.05 and 0.62 f 0.09 g, respectively) during the test meal. In both groups, serum gastrin concentration peaks 1 hr after the onset of feeding, with the hormone levels in diabetics being 2.2-fold greater than that found in normal mice (Figure 2). In addition, in diabetic animals, serum gastrin concentration remains at this high postprandial level for at least an additional two hr. In contrast, the hormone levels of normal mice abruptly decrease to below fasting values by the second postprandial hour. The difference in serum gastrin concentration between groups is significant at all time periods tested after feeding. The integrated gastrin secretory response to a meal was then approximated by integrating the area under the curve, when the data points from Figure 2 are plotted on linear graph paper. Using this technique, it was determined that the integrated gastrin response is 3.64fold greater in diabetic than normal mice over a 3-hr postprandial period. Molecular

Forms of Gastrin

In the next experiment, we investigated whether normal and diabetic animals had similar or constrasting molecular forms of gastrin. The results shown in Figure 3 convincingly demonstrate that the elution positions of gastrin immunoreactivity are comparable among the two groups of animals, with two prominent peaks being localized at 62-63% and 67-68X of the elution volume. The human ‘““I G-171 reference standard reproducibly eluted behind this region at 71-72%. This discrepancy between the elu-

Table 2. G-Cell Number” in Ad Libitum and Pair Fed Normal

G-Cell Number The number of gastrin immunoreactive Gcells for a given mucosal length was quantitated in the two groups of ad libitum fed mice. The results shown in Table 2 demonstrate that there is a modest,

Group Normal Diabetic

and Diabetic

Mice

Ad libitum fed 32.3 f 2.8 (9)h 42.1 f 2.5 (9)h

Pair fed 38.8 f 2.4 (8)b 38.6 + 3.9 (6)h

“G-cell number expressed as mean number of immunoreactive cells/mm mucosa + SEM. b ( ) = number of mice/group.

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LiCilTENRERGER

n

GASTROENTEROLOGY

AND RAMASWAMY

Vol. 77, No. 6

Normal

160-

Figure

l-

0

1 THE

AFTER ONSET

2. Gastrin secretory response to a I-hr test meal of chow in normal and genetically diabetic mice (4-5 mice/group).

2

OF FEEDING

(hrs)

tion positions of human and murine gastrin has been reported previously32.33 and probably indicates that rodents have variant molecular species of G-171 and G-1711 than that described for humans little gastrin. Discussion The experiments presented above clearly demonstrate that both serum and antral gastrin concentration are significantly elevated in genetically diabetic mice over the levels measured in normal littermates of the same strain (C57 BL/KSJ). The twofold increase in antral gastrin concentration is most likely attributable to both a greater number of Gcells and a higher gastrin content per G-cell in the diabetic state. This conclusion is based on the fact that the diabetes-associated increase in the number of immunoreactive G-cells (1%fold) is considerably less than the increase in radioimmunoassayable gastrin concentration (2.05fold). This discrepancy, therefore, suggests that G-cells of diabetic mice have a higher gastrin immunoreactivity than G-cells of normal mice. This could be caused by one of two factors: (a) an increased number of gastrin molecules per cell; and (b) a shift in the molecular species of gastrin in diabetic animals to a more immunopotent form of the hormone. This latter possibility, however, appears doubtful since it was determined that gastrin extracts of antral mucosas of normal and diabetic mice have similar elution profiles as determined by Sephadex chromatography. Definitive proof of the identity of the molecular forms of gastrin in the antrum of normal and diabetic mice, however, will await more sophisticated biochemical analysis. A factor which may contribute to the elevation of serum and antral gastrin concentration in diabetic animals is the increased food intake which is a characteristic behavioral abnormality associated with the syndrome. In confirmation with reports in the literature, we demonstrated that diabetic animals

consumed nearly twice the amount of food daily ingested by normals. Reports from our as well as other laboratories have indicated that the maintenance of both serum and antral gastrin is directly related to the amount of solid food consumed orally, since both tissue and serum gastrin levels are markedly decreased when animals are either fasted” or fed exclusively by vein.34.35Conversely, conditions resulting in hyperphagia are frequently associated with increases in gastrin concentration above normal values.3s*s’In studies presented here, we have demonstrated that diabetes-associated hypergastrinemia cannot be attributed to an increase in food consumption, since serum gastrin concentrations were comparably raised above normal in both diabetic mice feeding ad libitum and those which had access to

0

20

40

60

80

100

% OF EL”TlON VOLUME BETWEEN ALBVMIN AND SALT

forms of gastrin in the antral mucosas of Figure 3. Molecular normal and genetically diabetic mice, as determined by G-50 Superfine Sephadex chromatography (1 X 100 cm column).

December 1979

only a normal ration of chow. It should, however, be noted that the difference in antral gastrin concentration between groups was decreased somewhat in the pair feeding study from that recorded in freely feeding animals. This dietary difference may be due to the finding that the diabetic induced G-cell hyperplasia seen in ad libitum fed mice does not occur when diabetic animals are placed on a restricted diet. The diabetes associated increase in G-cell number, therefore, may be a nonspecific effect of increased food consumption, whereas the increase in the cellular content of gastrin molecules may be a specific characteristic of the diabetic state. Another finding of interest was that diabetic mice have a markedly enhanced gastrin secretory response to meal. The most dramatic differences between groups were seen several hours after feeding, as the serum gastrin levels of diabetic mice remained elevated, whereas the hormone levels of normal animals declined to fasting values. Since both groups of animals consumed a comparable amount of chow during the feeding period, this diabetic-induced alteration in the gastrin secretory response to a meal could not be attributed to differences in food intake. The finding that the serum gastrin levels reach a higher peak value after stimulation suggests that the releasable pool of gastrin molecules may be greater than normal in diabetic animals. This would be consistent with our finding, discussed previously, which suggests an increased cellular gastrin content in the diabetic animals. The duration of postprandial hypergastrinemia can be determined by several factors. First, it may be influenced by the gastric emptying rate, since increased gastric retention will result in an increase in the time luminal stimulants of gastrin release will remain in contact with the G-cells resulting in a prolongation of gastrin secretory stimulation. In support of this possibility, it is well documented that the gastric emptying rate is retarded in a high percentage of diabetic patients,lV4 and that in certain cases, complete gastric atony has been reported.” To our knowledge, the gastric emptying rate has not been investigated in animal models of diabetes and future motility studies are needed to understand further the relationships between the gastric emptying rate and gastrin levels in genetically diabetic mice. The prolonged gastrin secretory response to a meal may also be caused by a defect in the mechanism responsible for inhibiting hormone release after a challenge. Gastrin release in response to a stimulus is known to be completely abolished by a decreased antral acidification.““~“” Consequently, capacity to secrete gastric acid may result in hypergastrinemia, as occurs in patients with pernicious anemia or atrophic gastritis. Several laboratories have reported that both basal and stimulated gastric

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LEVELS

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acid secretion are reduced in a significant percentage of diabetic patients.‘-” In addition, certain patients are achlorhydric, which is consistent with the abnormally high incidence of pernicious anemia within the diabetic population.” Thus, genetically diabetic mice may similarly have a diminished ability to secrete gastric acid and this may play a role in the pathophysiology of diabetes associated hypergastrinemia due to reduced antral acidification. It is presently uncertain whether the increased gastrin levels in diabetes may be important in the mediation of any of the gastrointestinal alterations associated with the syndrome. It is well established that gastrin trophically affects the gastrointestinal mucosas stimulating protein, RNA and DNA synthesis, as well as cell proliferation.” As alluded to earlier, several laboratories, including our own, have reported that intestinal mass and cell proliferation are increased in several animal models of diabetes including the db/db strain investigated in this stUdy.‘7-W1 In addition, it has been demonstrated that the diabetes-induced increase in intestinal growth cannot be solely attributed to increased food intake,” suggesting that other, possible humoral factors, may play a role in the process. The identity of this humoral agent is unknown, and its characterization is complicated by the finding that the serum and tissue levels of many hormones are altered in diabetes. Of those reported to be elevated, only glucagon has been implicated as a possible stimulant of gastrointestinal growth.42~43The importance of glucagon as a potential growth agent has been minimized by the finding of Lorenz-Meyer et al.” that chronic glucagon injections, in fact, induce intestinal atrophy in the rat. It is, therefore, possible that the elevations in gastrin levels reported here may play an important role in the mediation of the increase in gastrointestinal growth in diabetes. References 1. Katz LA, Spiro HA: Gastrointestinal manifestations of diabetes. N Engl J Med 275:1350-1360,1966 2. Marshak RH, Maklansky D: Diabetic gastrophy. Am J Dig Dis 9:366-370,1964 3. Howland WJ, Drinkard RV: Acute diabetic gastric atony gastroparesis diabetacorum. JAMA 185:214-218.1963 4. Dotevall G: Gastric emptying in diabetes mellitus. Acta Med Stand 170:423-429,196l 5. Malins JM, French JM: Diabetic diarrhea. Q J Med 26:487-480, 1957 6. Whalen GE, Soergel KH, Geenen JE: Diabetic diarrhea. Gastroenterology 56:1021-1032,1969 7. Choe WY, Shay H, Shuman DR: External pancreatic secretion in diabetes mellitus. Ann Intern Med 59:812-821,1963 8. Vacca JB, Henke WJ, Knight WA Jr: Exocrine pancreas in diabetes mellitus. Ann Intern Med 61:242-247,1964 9. Angervall L, Dotevall G, Lehrmann KE: The gastric mucosa in diabetes mellitus. Acta Med Stand 169:339-349,196l

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10. Bowen BD, Aron AH: Gastric secretion in diabetes mellitus: report of .tO diabetic patients who had diarrhea and achlorhydria. Arch Intern Med 37:674-684,1926 11. Marks IN, Shuman CR, Shay H: Gastric acid secretion in diabetes mellitus. Ann Intern Med 51:227-237,1959 12. Sundberg A, Gronberg A: Diabetes mellitus and pernicious anemia. Acta Med Stand 166:147-150,196O 13. Crane RK: An effect of alloxan diabetes on the active transport of sugars by a rat small intestine in vitro. Biochim Biophys Acta Commun 4:436-440,196l 14. Schedl HP, Wilson HD: Effects of diabetes on intestinal growth and hexose transport in the rat. Am J Physiol 220:1739-1745,197l 15. Ramaswamy K, Peterson MA: Transport of monosaccharides by the small intestine of genetically diabetic mice. Fed Proc 37~772, 1978 16. Younoszai MK, Schedl HP: Effects of diabetes on intestinal disaccharidase activity. J Lab Clin Med 79:579-586.1972 17. Olsen WA, Roger L: Jejunal sucrase activity in diabetic rats. J Lab Clin Med 77:838-842.1971 18. Jervis EL, Levin RS: Anatomic adaptation of the alimentary tract of the rat to hyperphagia of chronic alloxan diabetes. Nature (Lond) 210:391-393,1966 19. Lorenz-Meyer H, Thiel F, Menge H, et al: Structural and functional studies on the transformation of the intestinal mucosa in rats with experimental diabetes. Res Exp Med 170:89-99, 1977 20. Miller KL, Hanson W, Schedl HP, et al: Proliferation rate and transit time of mucosal cells in small intestine of the diabetic rat. Gastroenterology 73:1326-13321977 21. Walsh JH, Grossman MI: Gastrin. N Engl J Med 292:13241332,1975 22. Johnson LR: Gastrointestinal hormones and their functions. Ann Rev Physiol39:135-148,1977 23. Hummel KP, Dickle MM, Coleman DL: Diabetes, a new mutation in the mouse. Science 153:1127-1128,1966 24. Herberg L, Coleman DL: Laboratory animals exhibiting obesity and diabetes syndromes. Metabolism 26:59-99,1977 25. Yalow RS, Berson SA: Radioimmunoassay of gastrin. Gastroenterology 58:1-14,197O 26. Walsh JH: Radioimmunoassay of gastrin. In: Nuclear Medicine In Vitro. Edited by B Rothfield. Philadelphia, J.B. Lippincott Co., pp 231-248,1974 27. Lichtenberger LM, Lechago J, Johnson LR: Depression of antral and serum gastrin concentration by food deprivation in the rat. Gastroenterology 68:1473-1479,1975 28. Dockray GJ, Walsh JH: Amino terminal gastrin in serum of Zollinger-Ellison Syndrome patients. Gastroenterology 68:222-230,1975

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29. Stadil FR, Rehfeld RF: Preparation of lZ51 labelled synthetic gastrin I for radioimmunoanalysis. Stand J Clin Invest 30:361-368,1972 30. Mason TE, Phifer RF, Spicer SS; An immunoglobulin-enzyme bridge method for localizing tissue antigens. J Histochem Cytochem 17:563-569,1969 31. Lloyd JB, Whalan WJ: An improved method for enzymic determination of glucose in the presence of maltose. Anal Biothem 30~467-470.1969 32. Holmquist AL, Walsh JH, Feldman EJ: Immunochemical distinction between rat gastrin and other mammalian gastrins. Gastroenterology 72:1071,1977 33. Lichtenberger LM, Shorey JM, Trier JS: Organ culture studies of rat antrum: Evidence for an antral inhibitor of gastrin release. Am J Physiol4:E410-E415,1978 34. Johnson LR, Copeland EM, Dudrick SJ, et al: Structural and hormonal alterations in the gastrointestinal tract of parenterally fed rats. Gastroenterology 68:1177-1183.1975 35. Johnson LR, Lichtenberger LM, Copeland EM, et al: Action of gastrin on gastrointestinal structure and function. Gastroenterology 68:1184-1192,1975 36. Lichtenberger LM, Johnson LR: A possible role of gastrin in the ontogenic development of the small intestine. Am J Physiol22?390-395.1974 37. Lichtenberger LM, Nance DM, Gorsk RA: Sex related differences in antral and serum gastrin levels in the rat. Proc Sot Exp Biol Med 151:785-788.1976 38. Debas HT, Walsh JH, Grossman MI: Mechanisms of release of antral gastrin. In: Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, The University of Texas Press, 425-435,1975 39. Walsh JH, Richardson CT, Fordtram HS: pH Dependence of acid secretion and gastrin release in normal and ulcer subjects. J Clin Invest 55:462-468,1975 40. Johnson LR: The trophic action of gastrointestinal hormones. Gastroenterology 70:278-288.1976 41. Lichtenberger LM, Ramaswamy K: Relationship between testinal mass and gastrin levels in normal and genetically betic mice (abstr). Gastroenterology 74:1056, 1978

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42. Laube H, Fussganger RD. Meier V: Hyperglucagonemia of the isolated perfused pancreas of the diabetic mice (db/db). Diabetologia 9:400-402,1973 43. Gleeson MH, Bloom SR, Polak JM, et al: An endocrine tumor in kidney affecting small bowel structure, motility, and absorptive function. Gut 12:733-742,197l 44. Lorenz-Meyer H, Menge H, Riecken EO: Functional and morphological studies on intestinal mucosa of the rat under chronic glucagon application. Res Exp Med 170:181-192,1977