The effect of insulin on delayed augmented mast-cell mediated mitogenesis in diabetic rats

Delayed augmented mitogenic reactivity follows mast-cell secretion in mesentery and skin in streptozotocin-diabetic rats with 4-week insulin-deficiency (Norrby 1982; Norrby et al. 1982; Norrby 1983). In such rats the basal proliferation is essentially unchanged in the mesentery and skin, whereas it is significantly increased in the small gut and significantly decreased in the kidney. On treating rats with 4-week-old diabetes with very-long-acting insulin for over 3 days (16 U/kg X 2 daily) the basal proliferation of the small gut and the kidney apparently became normal, the body weight increased, and the blood glucose concentration dropped substantially and progressively. However, insulin-treatment did not affect the mast-cell-dependent mitogenesis in the mesentery following intraperitoneal injection of the mast-cell secretagogue compound 48/80, as judged by specific DNA activity and mitosis counting. We conclude that some metabolic or cellular feature of diabetes which is not restored by 3-day insulin treatment, and not insulin deficiency itself, is the cause of the delayed increased mitogenic reactivity that follows mast-cell secretion in diabetic rat.     

Acute hyperglycaemia and mast-cell-dependent mitogenesis in rat

Hyperglycaemia is considered to be of possible aetiological significance for proliferative lesions in diabetes. We recently found that mast-cell-mediated proliferation in mesentery and skin is augmented in hyperglycaemic rats with longstanding diabetes. We now report a study to examine whether acute hyperglycaemia affects the proliferative response of fibroblasts and mesothelial cells that follows local mast-cell secretion in healthy rats. Hyperglycaemia was achieved by continuous intravenous infusion of glucose over a period of 2.5 h preceding and 9.5 h following administration of the mast-cell secretagogue compound 48/80. The 48/80 was given intraperitoneally which causes rapid mast-cell secretion and prompt subsequent mesenteric mitogenic stimulation. Hyperglycaemia thus persisted throughout the prereplicative period, during which stimulated cells are recruited into the cell cycle from a quiescent state. Because the ensuing mesenteric mitogenic response, estimated by specific DNA activity and mitosis counting, was unaffected by glucose infusion, acute hyperglycaemia itself appears to be of no aetiological significance for the accelerated mast-cell-dependent mesenteric mitogenesis in the diabetic rat.     

Delayed mast cell mediated mitogenic reactivity in diabetic rats

Rats with diabetes of 4 weeks' duration have previously demonstrated increased mitogenesis in normal connective tissue cells following mast cell secretion. The appearance of this augmented mast cell-mediated mitogenic reactivity was studied in the mesentery of insulin-deficient rats, 3, 7, and 28 days after they had been rendered diabetic by a single dose of streptozotocin. On day 7 mast cell secretion induced a subnormal mitogenic response which, however, increased above normal on day 28. This time lag in the augmentation of mitogenic responsiveness may be important since proliferative lesions in diverse mesenchymal tissues typically develop with a similar delay in both experimental and clinical diabetes.     

On disturbance of homeostasis in organ-cultured tissue

Summary The intact membranous rat mesentery was cultured in Eagle's minimum essential medium containing no serum or only low concentrations of serum. The procedure is in some important respects superior to previous organ culture techniques. To estimate the extent of disturbance of homeostasis of the tissue in culture, the spontaneous mast-cell histamine release was quantitated after preculture preparation of the specimens and after different intervals in culture. Also, the proliferation of fibroblasts and mesothelial cells that predominate in the mesentery was assessed at 48 h by cytofluorometric quantitation of DNA in single-tissue cells. Spontaneous histamine release was time dependent during cultivation, amounting to ca. 50% at 48 h, and was affected by the medium used for moistening the tissue before cultivation. Culturing also brought about great spontaneous increase in the proliferation of fibroblasts and mesothelial cells, the rate being related to the concentration of serum. Addition of the mast-cell secretagogues 48/80 or polymyxin B at 1 h caused rapid release of 50 to 60% of the histamine and was followed by augmented proliferation in the serum-containing media. The spontaneous increase of cell proliferation in tissue culture may be causally related to mast-cell secretion. Further studies are needed to define factors influencing the spontaneous mast-cell secretion and the mast-cell-dependent mitogenesis in normal tissue cells Supported by grants from the Swedish Medical Research Council (Project 5942) and State Board for Animal Experiments.     

Evidence of an acquired increase in mitogenesis in streptozotocin-diabetic rats, apparently relating to some tissue factor

We have previously reported that rats which have been suffering from streptozotocin-diabetes for 4 weeks show a supranormal mast cell mediated mitogenesis in mesenteric windows and in the skin; this late emerging, augmented mitogenic responsiveness appears, to be unaffected by insulin per se. To test whether this increased proliferogenic response is effected by some acquired quality within the tissue rather than a systemic factor in the blood, we studied mast cell mediated mitogenesis in organ-cultured intact mesenteric windows from rats with diabetes of 4 weeks' duration, using a biochemically-defined serum-free growth medium. Mast cells were activated by Compound 48/80 and their secretion was quantified biochemically in terms of histamine release. The mast cell-dependent mitogenic reaction in the predominant, morphologically discrete fibroblasts and mesothelial cells was quantified photometrically using Feulgen-absorption analysis of individual cell nuclei, and by determination of the mitotic index. Both types of target cell responded to a significantly greater degree mitogenically in diabetic compared with control tissue. This finding suggests that a considerable part of the increased mitogenic responsiveness previously observed in diabetic animals in vivo is causally related to some tissue-bound, i.e., cellular and/or extracellular factor(s) acquired during the course of the disease.     

Changes in LH and prolactin levels in diabetic male rats and the role of the opiate system in the control of their secretion

Diabetic male rat has low serum levels of luteinizing hormone (LH) and testosterone (T), which are accompanied by atrophy of the testes and accessory glands. The present study investigated changes in the serum levels of LH, prolactin (PRL) and glucose, following diabetes induction by streptozotocin. In addition, involvement of the opiate system in the control of LH and PRL secretion was evaluated. There was no difference in PRL levels between diabetic and control animals, except at 8 hours after streptozotocin injection. In contrast, the diabetic animals had consistently lower levels of LH, starting on the second day of diabetes. Blockade of the opiate system by naltrexone caused a sharp increase of LH levels in normoglycemic rats, while only a gradual decrease was observed in hyperglycemic animals. PRL secretion was inhibited by naltrexone, both in diabetic and control groups. It is concluded that, unlike normoglycemic rats, inhibition of LH secretion in diabetes is not under the control of the opiate system, probably as a result of T deficiency. In contrast, PRL secretion in diabetic rats, as in the control group, is under the influence of endogenous opiates.     

Insulin-dependent diabetes impairs the inflammatory response and delays angiogenesis following Achilles tendon injury

Although impaired wound healing associated with type 1 diabetes mellitus has been well studied in skin tissue, the influence of this metabolic disorder on tendon healing and recovery has not been extensively investigated. Because tendons are known to have limited repair potential, we studied the tendon-healing process by using a diabetic rat tendonitis model. We tested the hypothesis that diabetes influences the inflammatory response, cell proliferation, and angiogenesis in injured Achilles tendons. Diabetes was induced by injecting streptozotocin at 45 mg/kg body wt. Non-diabetic rats as well as diabetic and insulin-treated diabetic animals were then injected with collagenase. The accumulation of inflammatory cells was quantified in transversal sections of Achilles tendon by using immunohistochemical staining at days 0, 1, 3, 7, 14, and 28 posttrauma. The number of proliferative cells and the extent of neovascularization was also quantified in the paratenon and the core of the tendon at days 0, 3, 7, 14, and 28 posttrauma. Relative to nondiabetic and insulin-treated diabetic animals, the numbers of accumulated neutrophils and ED1(+) and ED2(+) macrophages in diabetic rats decreased by 46, 43, and 52%, respectively, in the first 3 days after injury compared with levels in nondiabetic and insulin-treated diabetic animals. The density of newly formed blood vessels decreased by 35 and 29% in the paratenon and the core of tendon, respectively, at days 3 and 7 after injury. Lastly, the concentration of proliferative cells decreased by 34% in the paratenon at day 7 posttrauma in injured tendons from diabetic rats relative to nondiabetic rats. These results indicate that alterations in inflammatory, angiogenic, and proliferative processes occurred in the diabetic state that might eventually perturb tendon healing and remodeling.     

On the 48÷80-induced secretion of tissue mast cells and its mitogenic effect on nearby cells in the intact rat

Histamine release from tissue-bound mast cells and cell proliferation in the proper mesentery in the intact rat was quantitated following in intraperitoneal injection of graded doses of compound 48/80. The dose-response curves were sigmoid-like in linear-log plots. ED50 for histamine release was 0.035-0.040 and for increased cell proliferation 0.040-0.048 microgram per g BW. The proliferative response following mast-cell secretion ceased after a period of between 48-72 h, irrespective of whether a high or a low dose of 48/80 was used. Basal on the net rate of histamine synthesis (ca. 0.45 microgram/g mesentery wet weight/h) after an initial injection of 48/80, on the extent of histamine release and the proliferative response after a repeated injection of 48/80, it is concluded that there is a lag period of at least 3 days before proliferation can be re-stimulated by renewed 48/80-induced mast-cell secretion.     

Normal mitotic reactivity of fibroblasts and mesothelial cells in thrombocytopenic rat

Mitotic reactivity following 48/80-induced mast-cell secretion was studied in the mesentery of rats made thrombocytopenic, 7 days following a single injection of melphalan. In spite of a low platelet count (7% of normal), the mitogenic reaction of the mesenteric fibroblasts and mesothelial cells was normal as judged by DNA-synthesis and mitotic index. The findings suggest that platelets and platelet-growth factors are not essential for the mast-cell-mediated mitogenic reaction of these two types of connective-tissue cells studied in vivo.     

A Novel Peptide with Similar Pharmacology to Exenatide in Rodents as GLP-1 Receptor Agonist

The prevalence of type-2 diabetes is rapidly increasing. Currently, exenatide is the first medicine which mimics incretin. However, it requires subcutaneous injection twice a day, an inconvenient way for patients. In this study, we identified a novel peptide with similar pharmacology to exenatide in rodents as GLP-1 receptor agonist which consists of 17 amino acids (17P). It promotes Ins-1 cell proliferation and insulin secretion and lowers blood glucose of diabetic rats. 17P was synthetized by solid-phase peptide synthesis. Interactions between GLP-1 receptor and 17P were studied by Bio-layer interferometry. Ins-1 cell proliferation was studied by MTT assay. ELISA was used to study Ins-1 cell insulin secretion. In vivo tests were performed with male Wistar rats. We used high fat diet and STZ injection to induce a type-2 diabetic rats model. Then, those rats were randomized to different test groups, and administered exenatide, 17P and saline water injection, to evaluate different responses. Based on HPLC (high performance liquid chromatography) and MS (mass spectrometry), 17P synthesis was successful. Bio-layer interferometry data showed a close interaction between GLP-1 receptor and 17P. 17P treatment of Ins-1 cells for 24 h could promote cell proliferation and insulin secretion in a dose-dependent manner. Administration of 17P in HF-STZ male Wistar rats demonstrated that 17P could lower the level of blood glucose and stabilize the body weight of the diabetic rats. All 17P treatments were similar to treatment with exenatide. In vitro and in vivo studies demonstrated that 17P could relieve symptoms of type-2 diabetes. Therefore, 17P could be developed as a promising type-2 diabetes therapeutic drug.     

Decreased NO signaling leads to enhanced vasoconstrictor responsiveness in skeletal muscle arterioles of the ZDF rat prior to overt diabetes and hypertension

Approximately 40% of patients with type 2 diabetes present with concurrent hypertension at the time of diabetes diagnosis. Increases in peripheral vascular resistance and correspondingly enhanced vasoconstrictor capacity could have profound implications for the development of hypertension and the progression of insulin resistance to overt diabetes. The purpose of this study was to determine whether skeletal muscle arteriolar vasoconstrictor dysfunction precedes or occurs concurrently with the onset of diabetes and hypertension. Male Zucker diabetic fatty (ZDF) rats were studied at 7, 13, and 20 wk of age to represent prediabetic and short-term and long-term diabetic states, respectively. Conscious mean arterial pressure (MAP), fasted plasma insulin and glucose, vasoconstrictor responses, and passive mechanical properties of isolated skeletal muscle arterioles were measured in prediabetic, diabetic, and age-matched control rats. Elevated MAP was manifest in short-term diabetes (control 117 +/- 1, diabetic 135 +/- 3 mmHg) and persisted with long-term diabetes (control 113 +/- 2, diabetic 135 +/- 3 mmHg). This higher MAP was preceded by augmented arteriolar vasoconstrictor responses to norepinephrine and endothelin-1 and followed by diminished beta-adrenergic vasodilation and enhanced myogenic constriction in long-term diabetes. Furthermore, we demonstrate that diminished nitric oxide (NO) signaling underlies the increases in vasoconstrictor responsiveness in arterioles from prediabetic and diabetic rats. Arteriolar stiffness was not different between control and prediabetic or diabetic rats at any time point studied. Collectively, these results indicate that increases in vasoconstrictor responsiveness resulting from diminished NO signaling in skeletal muscle arterioles precede the development of diabetes and hypertension in ZDF rats.     

Increased blood somatostatin concentration in streptozotocin diabetic rats

Immunoreactive somatostatin (IRS) was measured and characterized in plasma from the abdominal portel vein (PV) and inferior vena cava (IVC) of normal and streptozotocin diabetic rats. At 1 day following the induction of diabetes, PV and IVC levels of IRS were unchanged, at 2 weeks IVC IRS but not PV IRS was increased, but at 6 months both PV and IVC levels were strikingly elevated. Measurement of the IRS content of the pancreas and gut of the 6 month diabetic group showed a significant increase in the pancreas and stomach but not in the lower gut. Gel filtration of PV plasma from control and chronic diabetic rats revealed 3 immunoreactive forms with apparent m.w. of 12–15 K, 3.6 K and 1.6 K daltons. Immunoreactive somatostatin in the IVC was composed predominantly of the 12–15 K material in normal and diabetic rats. These results suggest that chronic but not acute streptozotocin diabetes leads to increased secretion of IRS from the pancreas and upper gut and to altered peripheral metabolism of IRS. Plasma IRS is heterogeneous, the main component being a high molecular weight form, the concentration of which is markedly increased in chronic insulinopenic diabetes.     

On the role of arachidonic acid metabolites in mast-cell mediated mitogenesis in the rat

We investigated whether the mitogenic response induced by local mast-cell secretion in the rat mesentery was affected by suppression of phospholipase A2, lipoxygenase, or cyclooxygenase in arachidonic acid metabolism. Enzyme inhibitor was given in a single intravenous dose 5 min before intraperitoneal injection of the mast-cell secretagogue 48/80. Mepacrine, a phospholipase A2 inhibitor, suppressed the generation of both leukotrienes (SRS) and prostaglandins (PG), whereas the lipoxygenase inhibitor BW 755C reduced the generation of SRS, and the cyclooxygenase inhibitor indomethacin significantly suppressed the generation of PG. None of the enzyme inhibitors affected the basal mesenteric histamine content or histamine release in the mesentery after exposure to 48/80, and none of them affected mast-cell-mediated mitogenesis in the mesentery as judged by specific DNA activity and mitosis counting. The stimulation of DNA synthesis and mitosis initiated by secreting mast cells is apparently not mediated or modulated by synthesis of leukotrienes, prostaglandins, or other known arachidonic acid metabolites.     

Insulin secretagogue effect of Ichnocarpus frutescence leaf extract in experimental diabetes: a dose-dependent study

Ichnocarpus frutescence (L.) R.Br. is an evergreen plant and many preparations have been used in traditional Indian medicine for centuries to treat several illnesses including diabetes. However, scientific evidence supporting these actions is lacking. In the present study we prepared various extracts of I. frutescence (IF) leaves which were tested against streptozotocin-induced diabetic rats. IF leaf methanolic extract (IFLMExt) showed significant plasma glucose lowering effect. Therefore, we prepared IFLMExt, which was tested against different types of glycemia (normal, glucose-fed hyperglycemic and streptozotocin-induced diabetic rats) for their potential to induce insulin secretion and cellular insulin responses. Fasting plasma glucose (FPG) levels were determined at different doses and times following treatment with IFLMExt or with vehicle in normal, glucose fed-hyperglycemic and diabetic rats. Oral administration of IFLMExt led to a significant blood glucose-lowering effect in glucose-fed hyperglycemic and diabetic rats. The hypoglycemic effect was observed at doses of 100 and 200 mg/(kg bw) after 6 and 2 h administration, respectively, in glucose-fed hyperglycemic rats. The maximum effect of IFLMExt was detected at 2 h with 200 mg/(kg bw) in diabetic animals and this profile was maintained for the next 6 h (37.23%) but increased after that at 24 h. Oral administration of IFLMExt daily for 45 days to diabetic rats significantly reduced the FPG (54.5%) to near normal. After 7 days of streptozotocin administration plasma insulin decreased in diabetic controls compared to normal controls. Treatment with IFLMExt significantly prevented the decrease in plasma insulin levels from day 0 to 45 in comparison to diabetic controls. Oral administration of n-hexane fraction led to a significant glucose-lowering effect in diabetic rats (54.50%). Histopathological examination showed that IFLMExt extract protected the pancreatic tissue from streptozotocin-induced damage enormously. Oral administration of IFLMExt extract and n-hexane fraction to normal and streptozotocin-induced diabetic rats decreased plasma glucose levels without hypoglycemic effect. The results suggest that methanolic extract and n-hexane fraction of IF may provide new therapeutic avenues against diabetes.     

Insulin release from the pancreas and fuel metabolism during late gestation in chemically diabetic rats

The effects of chemical diabetes and fasting on fuel metabolism and insulin secretory activity in late pregnancy were investigated. Female Wistar rats were made chemically diabetic (CD) by intravenous injection of streptozotocine (30 mg/kg) 2 weeks before conception. When CD pregnant rats were fed, plasma glucose and insulin levels were not significantly different from those of normal pregnant rats. Ketone body levels, however, were higher in CD pregnant rats than in normal pregnant rats, indicating insulin resistance in CD rats. Insulin secretion from the perfused pancreas caused by arginine or glucose was markedly decreased in CD pregnant rats. The pregnant rats were fasted for 2 days, from day 19 to 21 of gestation. Plasma glucose and insulin concentrations decreased similarly in the two groups, whereas ketone body concentrations in CD pregnant rats were significantly higher than those in normal pregnant rats. Glucose-induced insulin secretion by the perfused pancreas was markedly attenuated by fasting and was not significantly different in normal and CD pregnant rats. These observations suggest that diabetes mellitus accelerates starvation in late gestation, due to increased insulin resistance and poor insulin secretion, and that fasting in diabetic pregnancy amplifies ketogenesis.     

Influence of exercise on the immune function of rats of various ages

The purpose of this study was to determine whether exercise could prevent the age-related decline in mitogenesis, which has been well documented in rats, mice, and humans. At 1, 6, 12, and 18 mo of age, male Fischer F344 rats were subjected daily to swimming exercise for 6 mo. At the end of the 6-mo training period, spleen lymphocytes were isolated from the exercised rats and from age-matched sedentary controls. The induction of lymphocyte proliferation was measured with the mitogens concanavalin A (ConA) and lipopolysaccharide (LPS). In addition, the ability of the lymphocytes to produce interleukin 2 (IL 2) in response to ConA induction was measured. ConA- and LPS-induced proliferation decreased 41-63% between 7 and 25 mo of age in both exercised and sedentary control rats. ConA-induced IL 2 production decreased 42 and 62% between 7 and 25 mo of age for exercised and sedentary control rats, respectively. Although the age-related decline in mitogen-induced proliferation and IL 2 production was smaller in exercised rats, this was due to a lower level of mitogenesis and IL 2 production in lymphocytes from young exercised rats. Exercise resulted in a significant decrease (23-32%) in mitogen-induced lymphocyte proliferation and IL-2 production in 7-mo-old exercised rats compared with 7-mo-old sedentary rats. However, in the 18- and 24-mo-old rats, mitogen-induced lymphocyte proliferation and IL 2 production was not significantly different between exercised and sedentary control rats.     

Growth pattern switch of renal cells and expression of cell cycle related proteins at the early stage of diabetic nephropathy

Renal hypertrophy, partly due to cell proliferation and hypertrophy, has been found correlated to renal function deterioration in diabetes mellitus. We screened the up-regulated cell cycle related genes to investigate cell growth and the expression of cell cycle regulating proteins at the early stage of diabetic nephropathy using STZ-induced diabetic rats. Cyclin E, CDK(2) and P(27) were found significantly up-regulated in diabetic kidney. Increased cell proliferation in the kidney was seen at day 3, peaked at day 5, and returned to normal level at day 30. Cyclin E and CDK(2) expression also peeked at day 5 and P(27) activity peaked at day 14. These findings indicate that a hyperplastic growth period of renal cells is followed by a hypertrophic growth period at the early stage of diabetes. The growth pattern switch may be regulated by cell cycle regulating proteins, Cyclin E, CDK(2), and P(27).     

Distribution of vasoactive intestinal polypeptide, neuropeptide-Y and substance P and their effects on insulin secretion from the in vitro pancreas of normal and diabetic rats

This study examined the pattern of distribution of vasoactive intestinal polypeptide (VIP), neuropeptide-Y (NPY) and substance P (SP) in the pancreas of diabetic rat to determine whether there are changes in the number and pattern of distribution of these neuropeptides after the onset of diabetes. Moreover, the effect of VIP, NPY and SP on insulin secretion from the pancreas of normal and diabetic rats was also examined. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (i.p.) (60 mg kg body weight(-1)). Four weeks after the induction of DM, diabetic (n = 6) and normal (n = 6) rats were anesthetized with chloral hydrate and their pancreases removed and processed for immunohistochemistry and insulin secretion. The number of insulin-positive cells in the islets of Langerhans was reduced while that of VIP and NPY increased significantly after the onset of diabetes. The pattern of distribution of VIP, NPY and SP in the nerves innervating the pancreas was similar in both normal and diabetic rats. VIP-evoked large and significant (P < 0.02) increases in insulin secretion from the pancreas of normal and diabetic rats. NPY also induced a marked (P < 0.005) increase in insulin release from pancreatic tissue fragments of normal rat. Stimulation of pancreatic tissue fragments of diabetic rat with NPY resulted in a slight but not significant increase in insulin release. SP induced a large and significant (P < 0.005) increase in insulin secretion from the pancreas of normal rat but inhibited insulin secretion significantly (P < 0.03) from isolated pancreas of diabetic rat. In summary, VIP and NPY can stimulate insulin secretion from the pancreas after the onset of diabetes. The stimulatory effect of SP on insulin secretion is reversed to inhibitory in diabetic rats.     

Decreased cardiac output at the onset of diabetes: renal mechanisms and peripheral vasoconstriction

Recently we reported that hindquarter blood flow, measured 24 h/day, decreased progressively over the first 6 days of type 1 diabetes in rats. That response, coupled with the tendency of mean arterial pressure to increase, suggested a vasoconstrictor response. The purpose of this study was to measure the changes in cardiac output together with the renal hemodynamic and excretory responses to allow integrative determination of whether vasoconstriction likely accompanies the onset of type 1 diabetes. Rats were instrumented with a Transonic flow probe on the ascending aorta and with artery and vein catheters, and cardiac output and mean arterial pressure were measured continuously, 24 h/day, throughout the study. The induction of diabetes, by withdrawing intravenous insulin-replacement therapy in streptozotocin-treated rats, caused a progressive decrease in cardiac output that was 85 +/- 5% of control levels by day 7. This was associated with significant increases in glomerular filtration rate, renal blood flow, and microalbuminuria as well as urinary fluid and sodium losses, with a negative cumulative sodium balance averaging 15.7 +/- 1.6 meq by day 7. Restoring insulin-replacement therapy reversed the renal excretory responses but did not correct the negative sodium balance, yet cardiac output returned rapidly to control values. Increasing sodium intake during the diabetic and recovery periods also did not significantly affect the cardiac output response during any period. These results indicate that cardiac output decreases significantly at the onset of type 1 diabetes without glycemic control, and although volume loss may contribute to this response, there also is a component that is not volume or sodium dependent. We suggest this may be due to vasoconstriction, but to what extent local blood flow autoregulation or active vasoconstriction may have mediated that response is not known.     

Systemic Release of Mucosal Mast-cell Protease in Primed Brown Norway Rats after Feeding with β-Lactoglobulin

The plasma level of mucosal mast-cell protease was examined to find whether such measurements could be an indicator of allergic response to β-lactoglobulin (β-LG) challenged orally by rats. Brown Norway rats, which had been raised on a bovine milk-free diet, were systemically sensitized on day 0 with a low dose of β-LG, and then by an oral administration of β-LG for 3 h on day 14. The oral challenge with β-LG in saline, when compared to saline alone, resulted in a systemic elevation of rat mast-cell protease II (RMCPII), one of the specific markers for gut mucosal mast-cell secretion. The challenge with β-LG in a fat emulsion further increased the level of plasma RMCPII. This manipulation, however, was not successful for detecting any significant difference in mucosal leucotriene C₄, another allergic mediator. An oral challenge with polymerized β-LG did not induce any elevation of the protease, but resulted in a lower plasma level of β-LG-specific IgG. This animal model is thus relevant to investigate the events regulating the mucosal hypersensitivity and humoral immunity to food proteins.