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.     

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.     

Further studies on the delayed mast-cell-mediated mitogenesis in diabetic rat

We have investigated whether the delayed augmented mast-cell-dependent mitogenesis that we recently demonstrated in the true mesentery of diabetic rats may occur also in a tissue which is essentially different both functionally and structurally. Cutaneous proliferation in insulin-deficient rats following a single intradermal injection of compound 48/80 was assessed by the specific DNA activity and mitotic index, on days 7 and 28 after giving streptozotocin. The proliferation variables, which ran parallel, indicated that mast-cell secretion was not followed by proliferation on day 7, whereas there was markedly augmented proliferation on day 28. Mitosis counting showed that cell production following mast-cell secretion was significantly increased not only in the entire cutis but also in epithelial structures (epidermis and epidermal appendages). These findings are closely similar to the delayed augmentation of mast-cell-dependent mesenteric mitogenesis in diabetic rats. It is concluded that there may be a general pattern of delay in the appearance of augmented mast-cell-mediated mitogenesis in rats with chemically induced diabetes. It is questioned whether or not this delayed mitogenesis may be connected with the postponed proliferation in a variety of tissues and organs (such as arteries, eyes, and kidneys) that occurs in experimental and clinical diabetes.     

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 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.     

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.     

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.     

Amylin secretion from the perfused pancreas: dissociation from insulin and abnormal elevation in insulin-resistant diabetic rats

Amylin has been co-secreted from pancreatic islet beta-cells in constant proportion with insulin in some studies. We measured basal and glucose-stimulated amylin and insulin secretion from isolated perfused pancreases of normal and diabetic fatty Zucker rats. Glucose concentrations in the perfusion buffer were increased then decreased in small steps to mimic physiologic changes occurring after a meal. The absolute rate of amylin secretion and the molar ratio of amylin to insulin secreted from diabetic pancreases increased dramatically when infused glucose concentrations fell. Similar changes also occurred in normal pancreases, although the absolute change in amylin secretion was smaller. These studies provide the first evidence that (i) there is a mechanism within the pancreas whereby independent secretion of amylin and insulin can occur; (ii) the molar ratio of amylin to insulin secreted from both normal and diabetic pancreases can vary over a wide range; and (iii) there are important differences in the kinetics of amylin and insulin secretion or their coupling to stimulation by glucose between the isolated pancreases of normal rats and those with genetically transmitted insulin resistance and diabetes mellitus.     

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.     

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.     

LOCAL MITOGENIC EFFECT OF TISSUE MAST CELL SECRETION

The effect of drug-induced mast cell secretion on proliferation was studied in fibroblast-like and mesothelial-like cells in organ-cultured rat mesentery. Mast cell degranulation achieved by Compound 48/80 was followed by a marked mitogenic reaction in the surrounding tissue cells. The drug itself lacked mitogenic effect on cultured guinea-pig mesentery, the mast cells of which are unresponsive to the drug, and on a human normal fibroblast-like cell line. In contrast, histamine at about 10^?10 M, a major mast cell component, induced marked mitogenesis in guinea-pig mesentery without causing degranulation of mast cells. It is concluded that secreting rat-tissue mast cells release a mitogenic factor or factors acting locally on nearby tissue cells.     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

The CB1 Antagonist Rimonabant Decreases Insulin Hypersecretion in Rat Pancreatic Islets

Type 2 diabetes and obesity are characterized by elevated nocturnal circulating free fatty acids, elevated basal insulin secretion, and blunted glucose‐stimulated insulin secretion (GSIS). The CB1 receptor antagonist, Rimonabant, has been shown to improve glucose tolerance and insulin sensitivity in vivo but its direct effect on islets has been unclear. Islets from lean littermates and obese Zucker (ZF) and Zucker Diabetic Fatty (ZDF) rats were incubated for 24 h in vitro and exposed to 11 mmol/l glucose and 0.3 mmol/l palmitate (GL) with or without Rimonabant. Insulin secretion was determined at basal (3 mmol/l) or stimulatory (15 mmol/l) glucose concentrations. As expected, basal secretion was significantly elevated in islets from obese or GL‐treated lean rats whereas the fold increase in GSIS was diminished. Rimonabant decreased basal hypersecretion in islets from obese rats and GL‐treated lean rats without decreasing the fold increase in GSIS. However, it decreased GSIS in islets from lean rats without affecting basal secretion. These findings indicate that Rimonabant has direct effects on islets to reduce insulin secretion when secretion is elevated above normal levels by diet or in obesity. In contrast, it appears to decrease stimulated secretion in islets from lean animals but not in obese or GL‐exposed islets.     

Aluminum stimulates the proliferation and differentiation of osteoblasts in vitro by a mechanism that is different from fluoride

Summary Micromolar concentrations of aluminum sulfate consistently stimulated [³H]thymidine incorporation into DNA and increased cellular alkaline phosphatase activity (an osteoblastic differentiation marker) in osteoblast-line cells of chicken and human. The stimulations were highly reproducible, and were biphasic and dose-dependent with the maximal stimulatory dose varied from experiment to experiment. The mitogenic doses of aluminum ion also stimulated collagen synthesis in cultured human osteosarcoma TE-85 cells, suggesting that aluminum ion might stimulate bone formation in vitro. The effects of mitogenic doses of aluminum ion on basal osteocalcin secretion by normal human osteoblasts could not be determined since there was little, if any, basal secretion of osteocalcin by these cells. 1,25 Dihydroxyvitamin D₃ significantly stimulated the secretion of osteocalcin and the specific activity of cellular alkaline phosphatase in the human osteoblasts. Although mitogenic concentrations of aluminum ion potentiated the 1,25 dihydroxyvitamin D₃-dependent stimulation of osteocalcin secretion, they significantly inhibited the hormone-mediated activation of cellular alkaline phosphatase activity. Mitogenic concentrations of aluminum ion did not stimulate cAMP production in human osteosarcoma TE85 cells, indicating that the mechanism of aluminum ion does not involve cAMP. The mitogenic activity of aluminum ion is different from that of fluoride because (a) unlike fluoride, its mitogenic activity was unaffected by culture medium changes; (b) unlike fluoride, its mitogenic activity was nonspecific for bone cells; and (c) aluminum ion interacted with fluoride on the stimulation of the proliferation of osteoblastic-line cells, and did not share the same rate-limiting step(s) as that of fluoride. PTH interacted with and potentiated the bone cell mitogenic activity of aluminum ion, and thereby is consistent with the possibility that the in vivo osteogenic actions of aluminum ion might depend on PTH. In summary, low concentrations of aluminum ion could act directly on osteoblasts to stimulate their proliferation and differentiation by a mechanism that is different from fluoride.     

Feeding a Protective Hydrolysed Casein Diet to Young Diabetes-prone BB Rats Affects Oxidation of L[U?C14] glutamine in Islets and Peyer's Patches,Reduces Abnormally High Mitotic Activity in Mesenteric Lymph Nodes,Enhances Islet Insulin and Tends to Normalize NO Production

The present studies were undertaken to examine concomitant diet-induced changes in pancreatic islets and cells of the gut immune system of diabetes-prone BB rats in the period before classic insulitis. Diabetes-prone (BBdp) and control non-diabetes prone (BBc) BB rats were fed for ~ 17 days either a mainly plant-based standard laboratory rodent diet associated with high diabetes frequency, NIH-07 (NIH) or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. By about 7 weeks of age, NIH-fed BBdp rats had lower plasma insulin and insulin/glucose ratio, lower insulin content of isolated islets, lower basal levels of NO but higher responsiveness of NO production to IL-1β in cultured islets, and higher Con A response and biosynthetic activities in mesenteric lymphocytes than control rats fed the same diet. In control rats, the HC diet caused only minor changes in most variables, except for a decrease in oxidation of L-[U−C14]glutamine in Peyer''s patch (PP) cells and an increase in protein biosynthesis in mesenteric lymphocytes. In BBdp rats, however, the HC diet increased plasma insulin concentration, islet insulin/ protein ratio, and tended to normalize the basal and IL-1β-stimulated NO production by cultured islets. The HC diet decreased oxidation of L-[U−C14]glutamine in BBdp pancreatic islets, whereas oxidation of L-[U−C14]glutamine in PP cells was increased, and the basal [Methyl-H3] thymidine incorporation in mesenteric lymphocytes was decreased. These findings are compatible with the view that alteration of nutrient catabolism in islet cells as well as key cells of the gut immune system, particularly changes in mitotic and biosynthetic activities in mesenteric lymphocytes, as well as basal and IL-1β stimulated NO production, participate in the sequence of events leading to autoimmune diabetes in BB rats. Thus, the protection afforded by feeding a hydrolysed casein-based diet derives from alterations in both the target islet tissue and key cells of the gut immune system in this animal model of type 1 diabetes.     

Bile acid pool changes and regulation of cholate synthesis in experimental diabetes

The effect of alloxan-diabetes and insulin treatment in bile acid pool size and composition, bile acid secretion and cholic acid synthesis was investigated in the rat. The size of the cholate pool was significantly increased 4 days after diabetes induction. It reached a constant size three times that of control animals after 2 weeks of diabetes. Changes in bile acid pool size and secretion were directly dependent of the insulin deficiency state since they were reversed by insulin treatment and were not influenced by the caloric intake of the animal nor the pharmacologic effect of alloxan. Biliary cholate secretion was also 3-fold increased in diabetic rats and it accounted for more than 80% of the total bile acids compared to 60% in the control group. The calculated daily rate of cholate synthesis was increased in diabetic rats and the circadian rhythm of cholate synthesis was abolished in this condition. Therefore, it was shown that the negative feedback mechanism that regulates bile acid snythesis was deleted in diabetes. This mechanism was partially restored after 2 weeks of insulin treatment. These studies demonstrated that bile acid metabolism was profoundly changed in alloxan-diabetic rats and suggested that insulin may play an important role in the regulation of bile acid snythesis and intestinal absorption.     

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

Summary A mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/W ^vmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [³H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [³H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.Supported by a Grant-in-Aid for Scientific Research, No. 366, from the Japanese Ministry of Health and WelfareThe authors are indebted to Drs. Motomu Minamiyama and Yukio Hirata for valuable advices, and to Miss Mitsuko Inoue for technical assistance     

Follow-up of GK rats during prediabetes highlights increased insulin action and fat deposition despite low insulin secretion

The adult Goto-Kakizaki (GK) rat is characterized by impaired glucose-induced insulin secretion in vivo and in vitro, decreased beta-cell mass, decreased insulin sensitivity in the liver, and moderate insulin resistance in muscles and adipose tissue. GK rats do not exhibit basal hyperglycemia during the first 3 wk after birth and therefore could be considered prediabetic during this period. Our aim was to identify the initial pathophysiological changes occurring during the prediabetes period in this model of type 2 diabetes (T2DM). To address this, we investigated beta-cell function, insulin sensitivity, and body composition in normoglycemic prediabetic GK rats. Our results revealed that the in vivo secretory response of GK beta-cells to glucose is markedly reduced and the whole body insulin sensitivity is increased in the prediabetic GK rats in vivo. Moreover, the body composition of suckling GK rats is altered compared with age-matched Wistar rats, with an increase of the number of adipocytes before weaning despite a decreased body weight and lean mass in the GK rats. None of these changes appeared to be due to the postnatal nutritional environment of GK pups as demonstrated by cross-fostering GK pups with nondiabetic Wistar dams. In conclusion, in the GK model of T2DM, beta-cell dysfunction associated with increased insulin sensitivity and the alteration of body composition are proximal events that might contribute to the establishment of overt diabetes in adult GK rats.     

Effect of early dietary restriction on insulin action and secretion in the GK rat, a spontaneous model of NIDDM

The availability of the Goto-Kakisaki (GK) rat model of non-insulin-dependent diabetes mellitus prompted us to test the effect of a limited period of undernutrition in previously diabetic young rats on their insulin secretion and insulin action during adult age. Four-week-old female GK rats were either food restricted (35% restriction, 15% protein diet) or protein and energy restricted (35% restriction, 5% protein diet) for 4 wk. Food restriction in the young GK rat lowered weight gain but did not aggravate basal hyperglycemia or glucose intolerance, despite a decrease in basal plasma insulin level. Furthermore, the insulin-mediated glucose uptake by peripheral tissues in the GK rat was clearly improved. We also found that food restriction, when it is coupled to overt protein deficiency in the young GK rat, altered weight gain more severely and slightly decreased basal hyperglycemia but conversely aggravated glucose tolerance. Improvement of basal hyperglycemia was related to repression of basal hepatic glucose hyperproduction, despite profound attenuation of basal plasma insulin level. Deterioration of tolerance to glucose was related to severe blunting of the residual glucose-induced insulin secretion. It is, however, likely that the important enhancement of the insulin-mediated glucose uptake helped to limit the deterioration of glucose tolerance.