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Insulin and the gastrointestinal tract
Journal of Controlled Release 46 (1997) 89–98
Insulin and the gastrointestinal tract a, b c a Murray Saffran *, Ben Pansky , G. Colin Budd , Frederick E. Williams a
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43699 -0008, USA b Department of Anatomy and Neurobiology, Medical College of Ohio, Toledo, OH 43699 -0008, USA c Department of Physiology and Molecular Medicine, Medical College of Ohio, Toledo, OH 43699 -0008, USA Received 1 September 1995; accepted 24 April 1996
Abstract To provide a less cumbersome and more socially accepted form of insulin treatment than subcutaneous injections, we have designed an azopolymer system to deliver insulin with an absorption enhancer to the upper colon. In pancreatectomized dogs repeated oral doses of insulin in azopolymer-coated capsules lower the diabetic hyperglycemia to near normal values. Direct visualization of capsules containing radionuclides and coated with two new batches of azopolymer demonstrated that the capsules either passed intact through the gut or were opened in the small intestine. Direct visualization and insulin delivery using the same azopolymer will be necessary to locate the site of insulin delivery, but there remains the possibility that the insulin was delivered within the small intestine. To understand the effect of large doses of insulin delivered to the upper GI tract, an insulin solution was substituted for the drinking water of normal and diabetic rats. This produced temporary decreases in blood glucose levels, showing that in rats some absorption of insulin takes place above the colon. However, these rats became hyperphagic and lost weight. On post mortem examination the gut was distended with undigested food. Insulin seemed to inhibit processing of the food by the gut, in confirmation of observations by Elliasson and coworkers in human volunteers. The inhibitory effect of insulin on the gut, coupled with the presence of insulin receptors on the mucosal side of the gut and the presence of other pancreatic peptides in the gut, suggested to us that the gut may be able to make insulin. Accordingly, we looked for and found immunocytochemical evidence for preformed insulin in crypt cells in the colon and stomach, as well as the mRNAs for both rat insulins in similar cells. Gut insulin may be involved in the response of the gastrointestinal tract to food. Caution must be exerted in the introduction of high concentrations of insulin into the gastrointestinal tract. The same may be true of other powerful natural agents for which an oral delivery system would be desirable. Keywords: Oral insulin; Colon; Dogs; Rats; GI insulin
1. Motivation The main motivation for the work described in this *Corresponding author. Tel: 11-419-3814133; Fax: 11-4193895484; E-mail: [email protected].
paper is the often expressed desire of insulin-treated diabetic patients for a more convenient and socially compatible route of administration of insulin than subcutaneous injection. In addition, there has been some suspicion raised about the possibility that subcutaneous insulin may be at least partially respon-
0168-3659 / 97 / $17.00 Copyright 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0168-3659( 96 )01578-7
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sible for the cardiovascular complications ascribed to the disease itself [1]. Would the delivery of insulin by a more physiological route than subcutaneous injection be able to control the hyperglycemia without creating the climate for the cardiac and circulatory sequelae of long-term diabetes [2,3]?
include the mouth, the esophagus, the colon and the rectum. Of these, only the upper colon is drained almost completely into the hepatic portal circulation to mimic the route of physiological pancreatic insulin [5]. How can insulin be targeted to the colon if oral administration will route it through the dangers of the small intestine?
2. Physiological route Physiological insulin, made in the islets of the pancreas, is secreted directly into blood vessels that lead immediately into the hepatic portal circulation [2]. All the secreted insulin, therefore, acts in the first instance on the liver. Some of the insulin escapes binding and / or destruction in the liver [4] to pass into the general circulation to influence metabolic events in other tissues, notably the muscles and adipocytes. In contrast, insulin injected into a subcutaneous depot seeps into the general circulation, thereby exposing all tissues to an equal concentration of insulin; the liver receives only a fraction of the injected dose. The muscles and adipocytes can therefore respond to the injected dose without the insulin supply being subject to monitoring by the liver. The excessive exposure of the vasculature and other smooth muscles to injected insulin may set in train deleterious overstimulation of growth, cell division, and other metabolic responses that form the spectrum of diabetic complications [1]. To respond to the desires of insulin-treated diabetic patients and to provide insulin by a more physiologic route to the liver we embarked on a project to develop an orally effective delivery system for insulin.
4. Delivery to the colon The clue to the approach for the design of an oral colon delivery system was obtained from considering an anti-inflammatory drug used in treating the disease, ulcerative colitis. Ordinary anti-inflammatory agents administered orally will not reach the colon because they are mostly absorbed in the upper reaches of the gastrointestinal tract. However, the drug, salicylazosulfapyridine, variously known as Azulfidine or Salazopyrin, composed of an azo conjugate of 5-aminosalicylic acid and sulfapyridine, is insoluble in water and, therefore, passes through the small intestine unabsorbed. In the colon, the drug is split by bacterial reduction of the azo linkage into its components, 5-aminosalicylic acid and sulfapyridine (Fig. 1), to free the 5-aminosalicylic acid to act locally as an anti-inflammatory agent in the colon [6]. Could the immense reductive power of the
3. The plan I The dogma of insulin administration states that insulin cannot be taken orally because of two factors: (1) insulin is destroyed by digestive enzymes in the gut, and (2) insulin is too big a molecule to diffuse easily from the lumen of the gut into the blood. Most digestion of proteins and peptides occurs in the small intestine, with some proteolysis in the stomach. However, other parts of the gastrointestinal tract are relatively free of digestive activity. These
Fig. 1. Structure of salicylazosulfapyridine, a drug used in the management of ulcerative colitis, and its metabolism by colonic bacteria. The compound is split by the reduction of the azo bond (–N=N–) into aminosalicylic acid, an anti-inflammatory agent, and sulfapyridine, which is not anti-inflammatory.
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colonic flora be harnessed to deliver insulin to the colon? The colon is home to a very dense and diverse population of microorganisms. Estimates of the number of microorganisms in the colon contents are in the 10 11 –10 12 / g range [7]. The organisms feast on food residues and other matter delivered to the colon from the small intestine. Because of the density of cells in the lumen of the colon, little, if any, molecular oxygen survives to serve as a terminal electron acceptor in bacterial energy metabolism. Other electron sinks are pressed into service to create the most reducing environment of the body. Attesting to the reduction that takes place in the colon is the production of large amounts of almost fully reduced metabolic products, such as methane, fatty acids and hydrogen sulfide.
5. The plan II Could an impervious polymer be made that would (1) protect a drug load during the passage through the small intestine, (2) incorporate the elements of Azulfidine in its structure, (3) be degraded by colonic microorganisms sufficiently to expose the drug load in the colon for absorption into the blood leading to the hepatic portal vein?
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The criteria for successful candidate polymers were: (1) the polymer must be relatively impervious to water, and (2) the polymer must be susceptible to degradation by colonic bacteria. All samples were tested for ability to withstand the entry of water by coating a gelation capsule with the polymer. The capsule contained a mixture of sodium bicarbonate and salicylic acid. Entry of water generated carbon dioxide gas, which promptly blew up and sank the capsule. Only polymers that passed this test were then subjected to the next assay. To test for degradation by bacterial reduction, samples of the polymer were usually dissolved in chlorinated hydrocarbons and the solution was used to coat a metal grid. The solvent was thoroughly evaporated and the coated grids were placed into vessels containing a bacterial incubation medium seeded with samples of freshly voided human feces. The vessels were incubated anaerobically for up to a week. Because the bacterial density in the incubation vessels was much lower than in the colon, the incubation times were relatively long. The coated grids were removed at intervals from the incubation vessels, washed thoroughly with water, and examined in the scanning electron microscope. Bacterial degradation was denoted by the honeycomb appearance of the polymer surface (Fig. 2). Other tests of degradation were used, but the scanning electron micrograph was the best [9].
6. Implementation 7. First results in rats The task of designing and making such a polymer was assumed by Douglas C. Neckers, of the Department of Chemistry, Bowling Green State University, Bowling Green, Ohio, 0.5 h automobile ride from Toledo. Two of his young associates, Celine Savariar and G. Sudesh Kumar, first synthesized the 4,49divinylazobenzene reagents needed to make the polymer, and then successive forms of polymers, all including azo-linked chains in their structure [8]. More than 20 such polymers were made and sent for testing. The synthesis of the polymers was not trivial; too much cross-linking with divinylazobenzene and its derivatives produced products so insoluble as to be impossible to work with. Too little cross-linkage resulted in hydrogels that were incapable of protecting a drug load.
The concept was tested in rats, using vasopressin as a model peptide drug. Because of the small size of the rodent intestinal tract, ordinary capsules could not be used. Instead, tiny compressed tablets containing vasopressin (Innovative Research of America, Toledo, OH 43606, USA) were coated with azopolymer solutions; these were given orally to hydrated rats in an apparatus that automatically recorded urine formation. After a delay generated by the transit of the dosage form through the gastrointestinal tract, urine formation decreased abruptly as an indication of the delivery of the vasopressin load [10]. The vasopressin was delivered into the hepatic portal circulation, resulting in some first pass degradation of the vasopressin before it reached its target,
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Fig. 2. Scanning electron micrographs of metal grids coated with an azopolymer prepared by Sangekar et al. [15]. The grid on the left was incubated anaerobically for a week in medium alone. The surface is virtually unchanged by the incubation. The grid on the right was incubated in the same way except that the medium was innoculated with freshly voided human feces. The polymer film is honeycombed by the action of the bacteria, creating a porous structure.
the kidneys. Colonic delivery was more efficient that oral delivery of unprotected vasopressin, but less effective than rectal delivery, which mostly by-passes the liver [10]. Vasopressin dosage forms were also prepared by deposition of a vasopressin solution on tiny slivers of filter paper, which were then coated with azopolymers. In the next stage, similar dosage forms containing 1 unit each of insulin were prepared and given orally to rats. Here again delivery was effected after a delay. The insulin provoked decreases in the levels of glucose in the blood of rats made diabetic with streptozotocin [9].
8. Confirmation in dogs The record of attempts to give insulin by mouth is marked by successes or partial successes in rats, but by failures when attempts were made in larger animals, usually dogs. When a bolus of insulin is exposed in the gastrointestinal tract, absorption is dependent on passive diffusion because there is no known carrier system for the insulin molecule. Recent experiments have traced insulin molecules through the walls of the rat gastrointestinal tract into the blood, demonstrating that in spite of the large size of the molecules, they can penetrate the barriers [11]; the driving force is the concentration gradient
between the intestinal lumen and the blood. In the small diameter of the rodent colon, about 3–4 mm, the concentration of insulin and hence the lumenblood gradient can be high. However, the same dose of insulin released into the larger canine (diameter about 2 cm in beagles) or human colon (diameter of about 7.5 cm at the cecum) is diluted into a much larger volume, resulting in lower concentrations and consequently less absorption of insulin. We deemed it desirable to test our concept in a larger species than rats. To facilitate absorption of insulin from the larger colon, the absorption enhancer, 5-methoxysalicylate [12], would be added to the insulin in the capsule. In collaboration with James B. Field MD and his colleagues, then of the Department of Medicine at Baylor University, in Houston, Texas, dogs were made diabetic by removal of the pancreas. Cannulae and Doppler flow probes were placed in the hepatic portal vein and hepatic vein, to permit samples of blood entering the liver from the intestine and leaving the liver to be taken. Capsules of insulin (50–150 units per dose) and 5-methoxysalicylate (15–18 mg per dose), coated with azopolymers, were given to the dogs. At intervals insulin, glucose and glucagon-like immunoactivity were measured in plasma from blood samples taken from the cannulae. Evidence for the absorption of insulin from the intestine was obtained by a sudden rise in the insulin
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content of plasma from the cannula in the hepatic portal vein [13]. A smaller rise occurred in the blood leaving the liver. This was distinct evidence that insulin was absorbed from the orally administered dose. However, after a single dose, the decrease in glucose was small and did not last long. Glucagonlike immunoactivity, which originated in the gas-
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trointestinal tract in the pancreatectomized dog, decreased markedly. Because the glucose entering and leaving the liver was measured, glucose production by the liver could be calculated and was found to decrease to zero (and in some cases below zero — i.e. glucose uptake occurred) for a short time. In the virtual absence of an effect of the insulin dose on peripheral glucose utilization by muscle, the slight decrease in blood glucose is attributable to the combination of cessation of glucose production by the liver and continued glucose utilization by the brain. It became evident that a significant decrease in blood glucose to near normal levels could only be achieved if glucose production by the liver were inhibited over a longer period of time. Accordingly, oral administration of insulin capsules was repeated at 90-min intervals [13]. Now the mean production of glucose by the liver was kept below the rate of utilization by the brain and the blood level of glucose fell toward normal values (Fig. 3). In virtually all previous trials by many investigators of oral administration of insulin, only single doses were administered, with consequent small and short-lived decreases in blood glucose. Only when oral administration is repeated or prolonged, such as by a constant release from depots in the intestine [14], will the brain be able to use up the extra glucose, while the contribution of glucose to the blood by the liver is low or absent. The solution to successful oral administration of insulin is therefore to maintain a state of little or no gluconeogenesis in the liver to allow the brain to utilize blood glucose faster than it can be replenished. However, the consequences of prolonged inhibition of hepatic gluconeogenesis have yet to be determined.
9. Delivery site by azopolymers Fig. 3. Oral administration of 600 units of insulin and 5-methoxysalicylate in azopolymer coated gelatin capsules at intervals of 90 min to a pancreatectomized dog. The changes are shown in portal plasma insulin (upper left), arterial glucose (upper right), hepatic glucose production (middle left), portal plasma glucagon-like immunoactivity (middle right), and portal plasma flow (lower middle). The figure is modified from [13] and is reproduced with the permission of the Journal of Endocrinology Ltd.
Although the azopolymer coating of insulin capsules is designed to degrade only in the colon to deliver its load there, proof of the site of insulin delivery must be obtained by direct observation. Unfortunately the supply of azopolymer prepared by Kumar was exhausted. Accordingly, two new batches of polymer were made in another laboratory and
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were used to coat gelatin capsules containing gamma-emitting radionuclides to enable the capsules to be traced through the gastrointestinal tract and the site of delivery of the contents to be visualized [15]. One batch did not show any degradation by bacterial action, while the second did (Fig. 2). Capsules coated with the azopolymer that resisted bacterial action passed intact through the gastrointestinal tract of dogs. Capsules coated with the second batch that was degraded by bacteria opened in the region of the small intestine. Because the same azopolymers were not used, the observations with insulin delivery and direct visualization are not comparable. Until the same polymer can be used to deliver insulin and be visualized directly, there is the possibility that the insulin was actually delivered in the small intestine rather than the colon. Delivery in the small intestine would occur because the cross-linking of the azopolymer was insufficient to produce a truly impervious coat on the capsule, allowing water to enter prematurely. Too much cross-linking would produce a polymer that was resistant to bacterial action and would therefore pass intact through the entire gastrointestinal tract. More work must be done to produce a polymer with just the right characteristics to protect the load until the dosage form reaches the colon, where the coating can be degraded by bacterial reduction.
10. Evidence for insulin absorption Both immunocytochemical and immunochemical evidence for the transfer of insulin across the gut wall into the blood was obtained by the work of Bendayan and his colleagues [11]. Direct instillation of an insulin solution into various parts of the gastrointestinal tract was followed by visualization of insulin by immunocytochemical visualization with gold markers and immunoassay of insulin in the plasma. Gold particles were seen traversing the gut through enterocytes in all areas of the gastrointestinal tract. The efficiency of the transfer was estimated by the amount of insulin assayed in the plasma. The most efficient transfer followed instillation of insulin into the colon, where there is little if any digestion (Fig. 4).
Fig. 4. Plasma immunoreactive insulin in normal rats instilled directly into the duodenum or the colon with an insulin solution containing 2500 kallikrein inhibitory units of aprotinin, an inhibitor of proteolysis, and 10 mg of sodium cholate, an absorption enhancer. Drawn with the permission of the senior author from the data of Bendayan et al. [11].
11. Oral insulin solution With the possibility that insulin might have been delivered in the small intestine instead of the colon, and the observation that frequent doses of oral insulin can return the hyperglycemia of diabetes toward normal values, would insulin, dissolved in the drinking water at high concentrations, up to 100 units / ml, normalize the glycemia of diabetic rats? Because diabetic rats drink their body weight in water per 24 h, they would receive a continuous large dose of oral insulin. When such an experiment was performed on streptozotocin-diabetic rats, blood glucose levels fell by 5 to 10 mmol / l with a nadir at 2–3 h after replacing the drinking water with the insulin solution. The blood glucose levels began to climb again after about 48 h (Fig. 5). In non-diabetic rats, blood glucose also decreased by about 1.5 mmol / l, with a nadir at 2 h (Fig. 6). Unexpectedly, the normal and diabetic rats drinking insulin solution became hyperphagic, eating several times the number of food pellets as the control rats. Paradoxically, the insulin-treated rats lost body weight (Fig. 7)! On post-mortem examination, the upper gastrointestinal tracts of the insulin-treated rats were distended with undigested and unabsorbed food. The insulin treatment seemed to inhibit gastrointestinal motility, digestion and absorption. The rats were starving in spite of over-eating.
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Fig. 5. Blood glucose measurements in two fed streptozotocin diabetic rats drinking an insulin solution (100 units / ml5666 m mol / l) instead of water.
Fig. 6. Blood glucose measurements in two fed normal rats drinking an insulin solution (100 units / ml5666 m mol / l) instead of water.
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Fig. 8. A diagram of the effects of insulin in the liver, the muscles and the gastrointestinal tract. All contribute toward the limitation of hyperglycemia.
A recent experiment in Sweden with human volunteers, high parenteral doses of insulin, along with sufficient glucose to prevent hypoglycemia, induced significant decreases in the blood levels of the gastrointestinal peptide, motilin, which stimulates gastrointestinal motility. In these subjects both digestion of carbohydrate and absorption of glucose were inhibited [16]. These observations suggest that high concentrations of insulin reaching the intestine directly or via the blood inhibit the functions of the gastrointestinal tract. In the pancreatectomized dogs referred to earlier [13], oral insulin also decreased blood flow from the intestine to the liver, which may play a role in the decreased activity of the gut. All these effects of insulin would have the result of decreasing the glucose load of the body, while at the same time inhibiting gluconeogenesis by the liver and promoting glucose utilization by muscles (Fig. 8).
12. Insulin production by the gut
Fig. 7. Changes in body weight (line) and food intake (bars) of the same representative rat on drinking water or on insulin solution (100 units / ml5666 m mol / l). Mean insulin intake was 3.3 m mol / kg per h. The trial with insulin solution preceded that with water alone.
The existence of a gastroenteropancreatic axis has been known for many years [17]. The introduction of food into the gastrointestinal tract provokes the secretion not only of extrinsic pancreatic digestive enzymes (‘excretin’ effect), but also of insulin (‘incretin’ effect). Evidence points to the secretion by gastrointestinal cells of a product of the pre-
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proglucagon gene, glucagon-like peptide-1 (GLP-1), into the blood as the ‘incretin’, which sensitizes the pancreatic islet cells to receive the stimulus of an increase in blood glucose to secrete insulin [18]. Incretin, by itself, is ineffective as an insulin secretagogue. In our work with oral insulin in pancreatectomized dogs, we observed, as has been mentioned above [13], that oral insulin decreases the circulating levels of immunoreactive glucagon. Because of the absence of the pancreas, the most likely source of the immunoactivity is glucagon itself and / or GLP-1. Therefore, the control of secretion of glucagon / GLP1 in the gastrointestinal tract may be dual — glucose increases secretion, while insulin suppresses secretion. The source of enteral glucose is dietary carbohydrate. What, however, is the source of insulin in the gut? Insulin receptors have long been known to reside on the mucosal surface of cells in the gastrointestinal tract [19]. Where would insulin come from to bind to these receptors? A comparison of the peptide effectors found in the pancreas and gastrointestinal tract shows that the tissues contain a very similar list of peptides (Table 1). One peptide agent, insulin, is glaringly absent from the gastrointestinal tract, although it is the main peptide of pancreatic islets. Can the gut make insulin? Is gut insulin the source of the ligand for gut insulin receptors by a paracrine mechanism? Does gut insulin play a role in the control of the processing of food? Can the effects we observed when insulin was introduced into the gut be exaggerations of the physiological effects of insulin made in the gut? To answer these questions a search was made in gastrointestinal tissues, in collaboration with G.C. Budd, B. Pansky and K.S. Kendzierski for insulin and the cellular apparatus to make insulin in the gastrointestinal tract of the rat [20]. When sections of rat gut were exposed to conditions for the immunocytochemical identification of preformed insulin, staining was seen in scattered cells in the crypts of glandular structures in the stomach and colon, but not in the small intestine. Pancreatic sections from the same animals served as positive controls and stained only in the b cells of the islets. While this suggests that insulin may made by the gut, it might have arrived there from the blood and be bound to insulin receptors. Therefore, it was
necessary to demonstrate that the gut cells had the synthetic apparatus for making insulin. This involved the search for and identification of the mRNA for preproinsulin. mRNA for rat preproinsulin was located by in situ hybridization with labelled cDNA probes complementary to stretches of nucleotides known to be present in the mRNA for the two rat pancreatic insulins, rat insulin-1 and rat insulin-2. In addition, the mRNAs for the rat insulins were isolated from a mixture of mRNAs from rat colon, reverse transcribed to the corresponding DNAs, and amplified by PCR and then sequenced. The sequences were identical to those of the corresponding pancreatic nucleic acids. Treatment of the RNAs with restriction endonucleases produced the same pattern of fragments as with pancreatic samples. By both direct visualization of preformed insulin and the identification of the preproinsulin RNAs there is overwhelming evidence that the rat gut can indeed synthesize and store insulin. The insulin-forming cells have a similar distribution to the insulin-containing cells [20]. In view of our finding of insulin in the gastrointestinal tract, the observation by Bendayan et al. [11] of scattered ‘non-specific’ staining for insulin in control rat gastrointestinal tissue (i.e. not exposed to insulin solutions) may be of significance. How do these findings influence the long-held hope of developing an oral dosage form for insulin? We now recognize that introduction of insulin into the gastrointestinal tract may upset a physiological control system in which gastrointestinal insulin and insulin receptors play an important part. Only in the small intestine may an enteral insulin system be absent. Therefore, the best place to aim for enteral delivery of insulin may be in the small intestine, the segment most active in the digestive destruction of insulin. Until the distribution of insulin-forming gut cells and insulin receptors are completely mapped, parts of the gastrointestinal tract may be hazardous sites for the introduction of insulin. While this discouraging picture may be true for insulin, other peptides may be free of such complications. The introduction of powerful natural agents into parts of the intestine for absorption may not be harmless because of the wide variety of responding systems that may exist in this very complex organ. The presence of a very active immune system within the
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Table 1 Major regulatory peptides in the gut and the pancreas. The major pancreatic peptides are in bold type Gut [21]
Pancreas [22] Insulin
Products of the preproglucagon gene: Glicentin / Oxyntomodulin GLP-1 GLP-2 Somatostatin Gastrin Secretin CCK b -Lipotropin VIP GIP Substance P Neurotensin Motilin TRH GnRH Enkephalin Endorphins GRP PHI / PHM PYY NPY Galanin Pancreastatin Katacalcin Diazepam-binding inhibitor Calcitonin gene related peptide GRH CRH Neurokinins Neuromedins Valosin Head activator peptide Endothelin Vasoactive intestinal contractor
Glucagon
Somatostatin
CCK VIP GIP Substance P Neurotensin TRH Dynorphin Enkephalin Endorphins GRP PHI PYY NPY Galanin Pancreastatin Diazepam-binding inhibitor Calcitonin gene related peptide CRH
Islet amyloid polypeptide PP ANF Vasopressin
gut must also be considered whenever an effector is exposed to the gut surface. Oral toxicity testing must, therefore, be carried out routinely for every agent introduced into the gut for absorption. Insulin in the gastrointestinal tract may join the liver and the muscles in the body’s response to hypoglycemia. The gastrointestinal responses to insulin, namely a decreased blood flow from the intestine to the liver, decreased motility and de-
creased glucose absorption, all limit the flow of glucose into the blood, thereby contributing to the primary effect of insulin, the decrease in blood glucose (Fig. 8).
Acknowledgments Parts of the project were supported by grants from
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the National Institutes of Health, the Eli Lilly Company, the Diabetes Research and Education Foundation, the Lions Club and the Defiance Area Diabetes Club. A gift of human insulin from NovoNordisk, Copenhagen, is acknowledged. We thank Dr. Robert Trumbly for the use of his laboratory facilities. F.E.W. was supported by the Medical College of Ohio during the latter part of the work. Recent work was supported by grants from the Defiance Area Diabetes Club to Murray Saffran, G. Colin Budd, and Ben Pansky.
[11]
[12]
[13]
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Insulin and the gastrointestinal tract a, b c a Murray Saffran *, Ben Pansky , G. Colin Budd , Frederick E. Williams a
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43699 -0008, USA b Department of Anatomy and Neurobiology, Medical College of Ohio, Toledo, OH 43699 -0008, USA c Department of Physiology and Molecular Medicine, Medical College of Ohio, Toledo, OH 43699 -0008, USA Received 1 September 1995; accepted 24 April 1996
Abstract To provide a less cumbersome and more socially accepted form of insulin treatment than subcutaneous injections, we have designed an azopolymer system to deliver insulin with an absorption enhancer to the upper colon. In pancreatectomized dogs repeated oral doses of insulin in azopolymer-coated capsules lower the diabetic hyperglycemia to near normal values. Direct visualization of capsules containing radionuclides and coated with two new batches of azopolymer demonstrated that the capsules either passed intact through the gut or were opened in the small intestine. Direct visualization and insulin delivery using the same azopolymer will be necessary to locate the site of insulin delivery, but there remains the possibility that the insulin was delivered within the small intestine. To understand the effect of large doses of insulin delivered to the upper GI tract, an insulin solution was substituted for the drinking water of normal and diabetic rats. This produced temporary decreases in blood glucose levels, showing that in rats some absorption of insulin takes place above the colon. However, these rats became hyperphagic and lost weight. On post mortem examination the gut was distended with undigested food. Insulin seemed to inhibit processing of the food by the gut, in confirmation of observations by Elliasson and coworkers in human volunteers. The inhibitory effect of insulin on the gut, coupled with the presence of insulin receptors on the mucosal side of the gut and the presence of other pancreatic peptides in the gut, suggested to us that the gut may be able to make insulin. Accordingly, we looked for and found immunocytochemical evidence for preformed insulin in crypt cells in the colon and stomach, as well as the mRNAs for both rat insulins in similar cells. Gut insulin may be involved in the response of the gastrointestinal tract to food. Caution must be exerted in the introduction of high concentrations of insulin into the gastrointestinal tract. The same may be true of other powerful natural agents for which an oral delivery system would be desirable. Keywords: Oral insulin; Colon; Dogs; Rats; GI insulin
1. Motivation The main motivation for the work described in this *Corresponding author. Tel: 11-419-3814133; Fax: 11-4193895484; E-mail: [email protected].
paper is the often expressed desire of insulin-treated diabetic patients for a more convenient and socially compatible route of administration of insulin than subcutaneous injection. In addition, there has been some suspicion raised about the possibility that subcutaneous insulin may be at least partially respon-
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sible for the cardiovascular complications ascribed to the disease itself [1]. Would the delivery of insulin by a more physiological route than subcutaneous injection be able to control the hyperglycemia without creating the climate for the cardiac and circulatory sequelae of long-term diabetes [2,3]?
include the mouth, the esophagus, the colon and the rectum. Of these, only the upper colon is drained almost completely into the hepatic portal circulation to mimic the route of physiological pancreatic insulin [5]. How can insulin be targeted to the colon if oral administration will route it through the dangers of the small intestine?
2. Physiological route Physiological insulin, made in the islets of the pancreas, is secreted directly into blood vessels that lead immediately into the hepatic portal circulation [2]. All the secreted insulin, therefore, acts in the first instance on the liver. Some of the insulin escapes binding and / or destruction in the liver [4] to pass into the general circulation to influence metabolic events in other tissues, notably the muscles and adipocytes. In contrast, insulin injected into a subcutaneous depot seeps into the general circulation, thereby exposing all tissues to an equal concentration of insulin; the liver receives only a fraction of the injected dose. The muscles and adipocytes can therefore respond to the injected dose without the insulin supply being subject to monitoring by the liver. The excessive exposure of the vasculature and other smooth muscles to injected insulin may set in train deleterious overstimulation of growth, cell division, and other metabolic responses that form the spectrum of diabetic complications [1]. To respond to the desires of insulin-treated diabetic patients and to provide insulin by a more physiologic route to the liver we embarked on a project to develop an orally effective delivery system for insulin.
4. Delivery to the colon The clue to the approach for the design of an oral colon delivery system was obtained from considering an anti-inflammatory drug used in treating the disease, ulcerative colitis. Ordinary anti-inflammatory agents administered orally will not reach the colon because they are mostly absorbed in the upper reaches of the gastrointestinal tract. However, the drug, salicylazosulfapyridine, variously known as Azulfidine or Salazopyrin, composed of an azo conjugate of 5-aminosalicylic acid and sulfapyridine, is insoluble in water and, therefore, passes through the small intestine unabsorbed. In the colon, the drug is split by bacterial reduction of the azo linkage into its components, 5-aminosalicylic acid and sulfapyridine (Fig. 1), to free the 5-aminosalicylic acid to act locally as an anti-inflammatory agent in the colon [6]. Could the immense reductive power of the
3. The plan I The dogma of insulin administration states that insulin cannot be taken orally because of two factors: (1) insulin is destroyed by digestive enzymes in the gut, and (2) insulin is too big a molecule to diffuse easily from the lumen of the gut into the blood. Most digestion of proteins and peptides occurs in the small intestine, with some proteolysis in the stomach. However, other parts of the gastrointestinal tract are relatively free of digestive activity. These
Fig. 1. Structure of salicylazosulfapyridine, a drug used in the management of ulcerative colitis, and its metabolism by colonic bacteria. The compound is split by the reduction of the azo bond (–N=N–) into aminosalicylic acid, an anti-inflammatory agent, and sulfapyridine, which is not anti-inflammatory.
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colonic flora be harnessed to deliver insulin to the colon? The colon is home to a very dense and diverse population of microorganisms. Estimates of the number of microorganisms in the colon contents are in the 10 11 –10 12 / g range [7]. The organisms feast on food residues and other matter delivered to the colon from the small intestine. Because of the density of cells in the lumen of the colon, little, if any, molecular oxygen survives to serve as a terminal electron acceptor in bacterial energy metabolism. Other electron sinks are pressed into service to create the most reducing environment of the body. Attesting to the reduction that takes place in the colon is the production of large amounts of almost fully reduced metabolic products, such as methane, fatty acids and hydrogen sulfide.
5. The plan II Could an impervious polymer be made that would (1) protect a drug load during the passage through the small intestine, (2) incorporate the elements of Azulfidine in its structure, (3) be degraded by colonic microorganisms sufficiently to expose the drug load in the colon for absorption into the blood leading to the hepatic portal vein?
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The criteria for successful candidate polymers were: (1) the polymer must be relatively impervious to water, and (2) the polymer must be susceptible to degradation by colonic bacteria. All samples were tested for ability to withstand the entry of water by coating a gelation capsule with the polymer. The capsule contained a mixture of sodium bicarbonate and salicylic acid. Entry of water generated carbon dioxide gas, which promptly blew up and sank the capsule. Only polymers that passed this test were then subjected to the next assay. To test for degradation by bacterial reduction, samples of the polymer were usually dissolved in chlorinated hydrocarbons and the solution was used to coat a metal grid. The solvent was thoroughly evaporated and the coated grids were placed into vessels containing a bacterial incubation medium seeded with samples of freshly voided human feces. The vessels were incubated anaerobically for up to a week. Because the bacterial density in the incubation vessels was much lower than in the colon, the incubation times were relatively long. The coated grids were removed at intervals from the incubation vessels, washed thoroughly with water, and examined in the scanning electron microscope. Bacterial degradation was denoted by the honeycomb appearance of the polymer surface (Fig. 2). Other tests of degradation were used, but the scanning electron micrograph was the best [9].
6. Implementation 7. First results in rats The task of designing and making such a polymer was assumed by Douglas C. Neckers, of the Department of Chemistry, Bowling Green State University, Bowling Green, Ohio, 0.5 h automobile ride from Toledo. Two of his young associates, Celine Savariar and G. Sudesh Kumar, first synthesized the 4,49divinylazobenzene reagents needed to make the polymer, and then successive forms of polymers, all including azo-linked chains in their structure [8]. More than 20 such polymers were made and sent for testing. The synthesis of the polymers was not trivial; too much cross-linking with divinylazobenzene and its derivatives produced products so insoluble as to be impossible to work with. Too little cross-linkage resulted in hydrogels that were incapable of protecting a drug load.
The concept was tested in rats, using vasopressin as a model peptide drug. Because of the small size of the rodent intestinal tract, ordinary capsules could not be used. Instead, tiny compressed tablets containing vasopressin (Innovative Research of America, Toledo, OH 43606, USA) were coated with azopolymer solutions; these were given orally to hydrated rats in an apparatus that automatically recorded urine formation. After a delay generated by the transit of the dosage form through the gastrointestinal tract, urine formation decreased abruptly as an indication of the delivery of the vasopressin load [10]. The vasopressin was delivered into the hepatic portal circulation, resulting in some first pass degradation of the vasopressin before it reached its target,
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Fig. 2. Scanning electron micrographs of metal grids coated with an azopolymer prepared by Sangekar et al. [15]. The grid on the left was incubated anaerobically for a week in medium alone. The surface is virtually unchanged by the incubation. The grid on the right was incubated in the same way except that the medium was innoculated with freshly voided human feces. The polymer film is honeycombed by the action of the bacteria, creating a porous structure.
the kidneys. Colonic delivery was more efficient that oral delivery of unprotected vasopressin, but less effective than rectal delivery, which mostly by-passes the liver [10]. Vasopressin dosage forms were also prepared by deposition of a vasopressin solution on tiny slivers of filter paper, which were then coated with azopolymers. In the next stage, similar dosage forms containing 1 unit each of insulin were prepared and given orally to rats. Here again delivery was effected after a delay. The insulin provoked decreases in the levels of glucose in the blood of rats made diabetic with streptozotocin [9].
8. Confirmation in dogs The record of attempts to give insulin by mouth is marked by successes or partial successes in rats, but by failures when attempts were made in larger animals, usually dogs. When a bolus of insulin is exposed in the gastrointestinal tract, absorption is dependent on passive diffusion because there is no known carrier system for the insulin molecule. Recent experiments have traced insulin molecules through the walls of the rat gastrointestinal tract into the blood, demonstrating that in spite of the large size of the molecules, they can penetrate the barriers [11]; the driving force is the concentration gradient
between the intestinal lumen and the blood. In the small diameter of the rodent colon, about 3–4 mm, the concentration of insulin and hence the lumenblood gradient can be high. However, the same dose of insulin released into the larger canine (diameter about 2 cm in beagles) or human colon (diameter of about 7.5 cm at the cecum) is diluted into a much larger volume, resulting in lower concentrations and consequently less absorption of insulin. We deemed it desirable to test our concept in a larger species than rats. To facilitate absorption of insulin from the larger colon, the absorption enhancer, 5-methoxysalicylate [12], would be added to the insulin in the capsule. In collaboration with James B. Field MD and his colleagues, then of the Department of Medicine at Baylor University, in Houston, Texas, dogs were made diabetic by removal of the pancreas. Cannulae and Doppler flow probes were placed in the hepatic portal vein and hepatic vein, to permit samples of blood entering the liver from the intestine and leaving the liver to be taken. Capsules of insulin (50–150 units per dose) and 5-methoxysalicylate (15–18 mg per dose), coated with azopolymers, were given to the dogs. At intervals insulin, glucose and glucagon-like immunoactivity were measured in plasma from blood samples taken from the cannulae. Evidence for the absorption of insulin from the intestine was obtained by a sudden rise in the insulin
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content of plasma from the cannula in the hepatic portal vein [13]. A smaller rise occurred in the blood leaving the liver. This was distinct evidence that insulin was absorbed from the orally administered dose. However, after a single dose, the decrease in glucose was small and did not last long. Glucagonlike immunoactivity, which originated in the gas-
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trointestinal tract in the pancreatectomized dog, decreased markedly. Because the glucose entering and leaving the liver was measured, glucose production by the liver could be calculated and was found to decrease to zero (and in some cases below zero — i.e. glucose uptake occurred) for a short time. In the virtual absence of an effect of the insulin dose on peripheral glucose utilization by muscle, the slight decrease in blood glucose is attributable to the combination of cessation of glucose production by the liver and continued glucose utilization by the brain. It became evident that a significant decrease in blood glucose to near normal levels could only be achieved if glucose production by the liver were inhibited over a longer period of time. Accordingly, oral administration of insulin capsules was repeated at 90-min intervals [13]. Now the mean production of glucose by the liver was kept below the rate of utilization by the brain and the blood level of glucose fell toward normal values (Fig. 3). In virtually all previous trials by many investigators of oral administration of insulin, only single doses were administered, with consequent small and short-lived decreases in blood glucose. Only when oral administration is repeated or prolonged, such as by a constant release from depots in the intestine [14], will the brain be able to use up the extra glucose, while the contribution of glucose to the blood by the liver is low or absent. The solution to successful oral administration of insulin is therefore to maintain a state of little or no gluconeogenesis in the liver to allow the brain to utilize blood glucose faster than it can be replenished. However, the consequences of prolonged inhibition of hepatic gluconeogenesis have yet to be determined.
9. Delivery site by azopolymers Fig. 3. Oral administration of 600 units of insulin and 5-methoxysalicylate in azopolymer coated gelatin capsules at intervals of 90 min to a pancreatectomized dog. The changes are shown in portal plasma insulin (upper left), arterial glucose (upper right), hepatic glucose production (middle left), portal plasma glucagon-like immunoactivity (middle right), and portal plasma flow (lower middle). The figure is modified from [13] and is reproduced with the permission of the Journal of Endocrinology Ltd.
Although the azopolymer coating of insulin capsules is designed to degrade only in the colon to deliver its load there, proof of the site of insulin delivery must be obtained by direct observation. Unfortunately the supply of azopolymer prepared by Kumar was exhausted. Accordingly, two new batches of polymer were made in another laboratory and
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were used to coat gelatin capsules containing gamma-emitting radionuclides to enable the capsules to be traced through the gastrointestinal tract and the site of delivery of the contents to be visualized [15]. One batch did not show any degradation by bacterial action, while the second did (Fig. 2). Capsules coated with the azopolymer that resisted bacterial action passed intact through the gastrointestinal tract of dogs. Capsules coated with the second batch that was degraded by bacteria opened in the region of the small intestine. Because the same azopolymers were not used, the observations with insulin delivery and direct visualization are not comparable. Until the same polymer can be used to deliver insulin and be visualized directly, there is the possibility that the insulin was actually delivered in the small intestine rather than the colon. Delivery in the small intestine would occur because the cross-linking of the azopolymer was insufficient to produce a truly impervious coat on the capsule, allowing water to enter prematurely. Too much cross-linking would produce a polymer that was resistant to bacterial action and would therefore pass intact through the entire gastrointestinal tract. More work must be done to produce a polymer with just the right characteristics to protect the load until the dosage form reaches the colon, where the coating can be degraded by bacterial reduction.
10. Evidence for insulin absorption Both immunocytochemical and immunochemical evidence for the transfer of insulin across the gut wall into the blood was obtained by the work of Bendayan and his colleagues [11]. Direct instillation of an insulin solution into various parts of the gastrointestinal tract was followed by visualization of insulin by immunocytochemical visualization with gold markers and immunoassay of insulin in the plasma. Gold particles were seen traversing the gut through enterocytes in all areas of the gastrointestinal tract. The efficiency of the transfer was estimated by the amount of insulin assayed in the plasma. The most efficient transfer followed instillation of insulin into the colon, where there is little if any digestion (Fig. 4).
Fig. 4. Plasma immunoreactive insulin in normal rats instilled directly into the duodenum or the colon with an insulin solution containing 2500 kallikrein inhibitory units of aprotinin, an inhibitor of proteolysis, and 10 mg of sodium cholate, an absorption enhancer. Drawn with the permission of the senior author from the data of Bendayan et al. [11].
11. Oral insulin solution With the possibility that insulin might have been delivered in the small intestine instead of the colon, and the observation that frequent doses of oral insulin can return the hyperglycemia of diabetes toward normal values, would insulin, dissolved in the drinking water at high concentrations, up to 100 units / ml, normalize the glycemia of diabetic rats? Because diabetic rats drink their body weight in water per 24 h, they would receive a continuous large dose of oral insulin. When such an experiment was performed on streptozotocin-diabetic rats, blood glucose levels fell by 5 to 10 mmol / l with a nadir at 2–3 h after replacing the drinking water with the insulin solution. The blood glucose levels began to climb again after about 48 h (Fig. 5). In non-diabetic rats, blood glucose also decreased by about 1.5 mmol / l, with a nadir at 2 h (Fig. 6). Unexpectedly, the normal and diabetic rats drinking insulin solution became hyperphagic, eating several times the number of food pellets as the control rats. Paradoxically, the insulin-treated rats lost body weight (Fig. 7)! On post-mortem examination, the upper gastrointestinal tracts of the insulin-treated rats were distended with undigested and unabsorbed food. The insulin treatment seemed to inhibit gastrointestinal motility, digestion and absorption. The rats were starving in spite of over-eating.
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Fig. 5. Blood glucose measurements in two fed streptozotocin diabetic rats drinking an insulin solution (100 units / ml5666 m mol / l) instead of water.
Fig. 6. Blood glucose measurements in two fed normal rats drinking an insulin solution (100 units / ml5666 m mol / l) instead of water.
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Fig. 8. A diagram of the effects of insulin in the liver, the muscles and the gastrointestinal tract. All contribute toward the limitation of hyperglycemia.
A recent experiment in Sweden with human volunteers, high parenteral doses of insulin, along with sufficient glucose to prevent hypoglycemia, induced significant decreases in the blood levels of the gastrointestinal peptide, motilin, which stimulates gastrointestinal motility. In these subjects both digestion of carbohydrate and absorption of glucose were inhibited [16]. These observations suggest that high concentrations of insulin reaching the intestine directly or via the blood inhibit the functions of the gastrointestinal tract. In the pancreatectomized dogs referred to earlier [13], oral insulin also decreased blood flow from the intestine to the liver, which may play a role in the decreased activity of the gut. All these effects of insulin would have the result of decreasing the glucose load of the body, while at the same time inhibiting gluconeogenesis by the liver and promoting glucose utilization by muscles (Fig. 8).
12. Insulin production by the gut
Fig. 7. Changes in body weight (line) and food intake (bars) of the same representative rat on drinking water or on insulin solution (100 units / ml5666 m mol / l). Mean insulin intake was 3.3 m mol / kg per h. The trial with insulin solution preceded that with water alone.
The existence of a gastroenteropancreatic axis has been known for many years [17]. The introduction of food into the gastrointestinal tract provokes the secretion not only of extrinsic pancreatic digestive enzymes (‘excretin’ effect), but also of insulin (‘incretin’ effect). Evidence points to the secretion by gastrointestinal cells of a product of the pre-
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proglucagon gene, glucagon-like peptide-1 (GLP-1), into the blood as the ‘incretin’, which sensitizes the pancreatic islet cells to receive the stimulus of an increase in blood glucose to secrete insulin [18]. Incretin, by itself, is ineffective as an insulin secretagogue. In our work with oral insulin in pancreatectomized dogs, we observed, as has been mentioned above [13], that oral insulin decreases the circulating levels of immunoreactive glucagon. Because of the absence of the pancreas, the most likely source of the immunoactivity is glucagon itself and / or GLP-1. Therefore, the control of secretion of glucagon / GLP1 in the gastrointestinal tract may be dual — glucose increases secretion, while insulin suppresses secretion. The source of enteral glucose is dietary carbohydrate. What, however, is the source of insulin in the gut? Insulin receptors have long been known to reside on the mucosal surface of cells in the gastrointestinal tract [19]. Where would insulin come from to bind to these receptors? A comparison of the peptide effectors found in the pancreas and gastrointestinal tract shows that the tissues contain a very similar list of peptides (Table 1). One peptide agent, insulin, is glaringly absent from the gastrointestinal tract, although it is the main peptide of pancreatic islets. Can the gut make insulin? Is gut insulin the source of the ligand for gut insulin receptors by a paracrine mechanism? Does gut insulin play a role in the control of the processing of food? Can the effects we observed when insulin was introduced into the gut be exaggerations of the physiological effects of insulin made in the gut? To answer these questions a search was made in gastrointestinal tissues, in collaboration with G.C. Budd, B. Pansky and K.S. Kendzierski for insulin and the cellular apparatus to make insulin in the gastrointestinal tract of the rat [20]. When sections of rat gut were exposed to conditions for the immunocytochemical identification of preformed insulin, staining was seen in scattered cells in the crypts of glandular structures in the stomach and colon, but not in the small intestine. Pancreatic sections from the same animals served as positive controls and stained only in the b cells of the islets. While this suggests that insulin may made by the gut, it might have arrived there from the blood and be bound to insulin receptors. Therefore, it was
necessary to demonstrate that the gut cells had the synthetic apparatus for making insulin. This involved the search for and identification of the mRNA for preproinsulin. mRNA for rat preproinsulin was located by in situ hybridization with labelled cDNA probes complementary to stretches of nucleotides known to be present in the mRNA for the two rat pancreatic insulins, rat insulin-1 and rat insulin-2. In addition, the mRNAs for the rat insulins were isolated from a mixture of mRNAs from rat colon, reverse transcribed to the corresponding DNAs, and amplified by PCR and then sequenced. The sequences were identical to those of the corresponding pancreatic nucleic acids. Treatment of the RNAs with restriction endonucleases produced the same pattern of fragments as with pancreatic samples. By both direct visualization of preformed insulin and the identification of the preproinsulin RNAs there is overwhelming evidence that the rat gut can indeed synthesize and store insulin. The insulin-forming cells have a similar distribution to the insulin-containing cells [20]. In view of our finding of insulin in the gastrointestinal tract, the observation by Bendayan et al. [11] of scattered ‘non-specific’ staining for insulin in control rat gastrointestinal tissue (i.e. not exposed to insulin solutions) may be of significance. How do these findings influence the long-held hope of developing an oral dosage form for insulin? We now recognize that introduction of insulin into the gastrointestinal tract may upset a physiological control system in which gastrointestinal insulin and insulin receptors play an important part. Only in the small intestine may an enteral insulin system be absent. Therefore, the best place to aim for enteral delivery of insulin may be in the small intestine, the segment most active in the digestive destruction of insulin. Until the distribution of insulin-forming gut cells and insulin receptors are completely mapped, parts of the gastrointestinal tract may be hazardous sites for the introduction of insulin. While this discouraging picture may be true for insulin, other peptides may be free of such complications. The introduction of powerful natural agents into parts of the intestine for absorption may not be harmless because of the wide variety of responding systems that may exist in this very complex organ. The presence of a very active immune system within the
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Table 1 Major regulatory peptides in the gut and the pancreas. The major pancreatic peptides are in bold type Gut [21]
Pancreas [22] Insulin
Products of the preproglucagon gene: Glicentin / Oxyntomodulin GLP-1 GLP-2 Somatostatin Gastrin Secretin CCK b -Lipotropin VIP GIP Substance P Neurotensin Motilin TRH GnRH Enkephalin Endorphins GRP PHI / PHM PYY NPY Galanin Pancreastatin Katacalcin Diazepam-binding inhibitor Calcitonin gene related peptide GRH CRH Neurokinins Neuromedins Valosin Head activator peptide Endothelin Vasoactive intestinal contractor
Glucagon
Somatostatin
CCK VIP GIP Substance P Neurotensin TRH Dynorphin Enkephalin Endorphins GRP PHI PYY NPY Galanin Pancreastatin Diazepam-binding inhibitor Calcitonin gene related peptide CRH
Islet amyloid polypeptide PP ANF Vasopressin
gut must also be considered whenever an effector is exposed to the gut surface. Oral toxicity testing must, therefore, be carried out routinely for every agent introduced into the gut for absorption. Insulin in the gastrointestinal tract may join the liver and the muscles in the body’s response to hypoglycemia. The gastrointestinal responses to insulin, namely a decreased blood flow from the intestine to the liver, decreased motility and de-
creased glucose absorption, all limit the flow of glucose into the blood, thereby contributing to the primary effect of insulin, the decrease in blood glucose (Fig. 8).
Acknowledgments Parts of the project were supported by grants from
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the National Institutes of Health, the Eli Lilly Company, the Diabetes Research and Education Foundation, the Lions Club and the Defiance Area Diabetes Club. A gift of human insulin from NovoNordisk, Copenhagen, is acknowledged. We thank Dr. Robert Trumbly for the use of his laboratory facilities. F.E.W. was supported by the Medical College of Ohio during the latter part of the work. Recent work was supported by grants from the Defiance Area Diabetes Club to Murray Saffran, G. Colin Budd, and Ben Pansky.
[11]
[12]
[13]
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