Insulin-induced granuloma tissue formation and angiogenesis in alloxan-treated diabetic mice

Pouch granuloma formation induced by Freund's complete adjuvant containing 0.1% croton oil was studied in both normal and alloxan-induced diabetic mice, and was found to be significantly suppressed in the diabetic group. Insulin repeatedly injected into the pouch facilitated granuloma formation dose-dependently, especially in the diabetic mice. The topical insulin treatment did not affect blood glucose levels. The suppression of granuloma formation by diabetes and its reverse by insulin treatment were verified by histological findings in the granuloma pouch wall. Characteristic changes in neovascularization occurred in the pouch wall. The hydroxyproline content in the granuloma tissue in diabetic mice was not significantly enhanced by the insulin treatment. This indicates that differences in collagen production in normal and diabetic mice were not the critical factor affecting granuloma formation. It was concluded that the decreased granuloma formation in the diabetic state was due to the lack of insulin as a growth factor, and that angiogenesis induced by insulin preceding collagen fiber formation may play an essential role in granuloma formation.     

Diabetic state-induced modification of Ca, Mg, Fe and Zn content of skeletal, cardiac and smooth muscles

The metal content of diaphragm, gastrocnemius, ventricle, and bladder muscles in genetically obese diabetic KK-CAy and alloxan-diabetic ddY mice was compared with that in prediabetic KK-CAy and normal ddY mice, because the muscles of the diabetic KK-CAy mice had morphological abnormalities, such as atrophy, disappearance of Z-band, disturbed myofibrils and swollen sarcoplasmic reticulum. The amounts of calcium (Ca) in gastrocnemius, ventricle and bladder muscles from the prediabetic KK-CAy mice were significantly 7.7, 98.3, and 36.9% greater, respectively, than those in normal ddY mice. In contrast, the magnesium (Mg) content of the diaphragm, the gastrocnemius, and the ventricle in the prediabetic mice was 8.6, 7.4, and 4.3% lower, respectively, than in the ddY mice. The iron (Fe) content of the diaphragm, gastrocnemius, and ventricle muscles in the prediabetic mice was 29.2, 43.6, and 44.6% greater, respectively, than in the ddY mice. The Ca content in the gastrocnemius muscles of the diabetic KK-CAy mice and the alloxan-diabetic mice was 19.8 and 11.7% higher, respectively, than in the prediabetic and normal mice. The Ca content of the ventricle muscle was increased only in the alloxan-mice. The gastrocnemius Mg was also 9.0 and 5.5% greater in the KK-CAy and the alloxan-mice. The Fe content of the diaphragm and the gastrocnemius muscles from the KK-CAy mice was 27.3 and 23.2% greater, respectively, than in the prediabetic mice. The zinc (Zn) content of the gastrocnemius and the bladder was 16.4 and 18.0% higher, but the ventricle Zn was 13.4% lower, respectively, than in the prediabetic control. The changes in metal content induced by the diabetic state may be related to the morphological abnormalities.     

A genetically diabetic model "KK-CAy mice" for a pharmacological assay.

A genetically diabetic model, KK-CAy mice which were bred by mating female KK mice (aa, BB, cc) with male KK-CAy mice (Aya, BB, CC) was studied on the usefulness as a tool for a pharmacological assay. Body weights of KK-CAy mice increased more rapidly than those of control mice, KK-C. When the body weights of male KK-CAy mice reached about 30 g 10 weeks after birth, their blood glucose levels increased. Severe hyperglycemia (over 300 mg/100 ml) was often observed in the males, but not in the females. Glucose tolerance in the KK-CAy mice was more markedly impaired than that in the control mice. The increase in blood FFA level correlated with the increase in body weight on both KK-CAy mice and the controls. On hyperinsulinemia observed, the ratio of plasma immunoreactive insulin (IRI) level to blood glucose level in the male mice was lower than that seen in the female mice. On hyperglucagonemia observed, elevation of plasma immunoreactive glucagon (IRG) was more remarkable in the males than in the females. Morphological study showed insular degranulation only in the males. Since the dose-dependent insulin-induced falling was observed on blood glucose level in nonfasted KK-CAy mice, they could be used as a feasible tool for an assay of antidiabetic drugs.     

Effects of antidiabetic drugs on glucose tolerance in streptozotocin-nicotinamide-induced mildly diabetic and streptozotocin-induced severely diabetic mice

In this study, streptozotocin-nicotinamide-induced mildly diabetic mice and streptozotocin-induced severely diabetic mice were created to compare their characteristics and to investigate the effects of antidiabetic drugs on glucose tolerance. In severely diabetic mice, the pancreatic insulin content decreased to approximately 10% of levels found in normal mice. These mice also showed a decrease in body weight, a marked increase in nonfasting blood glucose levels and urinary glucose excretion, and a marked decline in glucose tolerance due to insulin secretory deficiency. In contrast, the pancreatic insulin content was approximately 50% of normal levels in mildly diabetic mice. These mice did not show any change in body weight, but displayed a mild increase in nonfasting blood glucose levels and urinary glucose excretion, and a mild decline in glucose tolerance due to loss of early-phase insulin secretion. Administration of antidiabetic drugs, namely voglibose, metformin, glibenclamide, sitagliptin and insulin, significantly improved glucose tolerance in mildly diabetic mice. In severely diabetic mice, voglibose, metformin and insulin significantly improved glucose tolerance, but no significant effect was observed for glibenclamide and sitagliptin due to a decreased insulinotropic effect. These results demonstrate that streptozotocin-nicotinamide-induced mildly diabetic mice have many pathological features resembling type 2 diabetes, and can serve as models for the pharmacological evaluation of many antidiabetic drugs.     

Polyamine and amino acid content, and activity of polyamine-synthesizing decarboxylases, in liver of streptozotocin-induced diabetic and insulin-treated diabetic rats

1. Concentrations of polyamines, amino acids, glycogen, nucleic acids and protein, and activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, were measured in livers from control, streptozotocin-diabetic and insulin-treated diabetic rats. 2. Total DNA per liver and protein per mg of DNA were unaffected by diabetes, whereas RNA per mg of DNA and glycogen per g of liver were decreased. Insulin treatment of diabetic rats induced both hypertrophy and hyperplasia, as indicated by an increase in all four of these constituents to or above control values. 3. Spermidine content was increased in the livers of diabetic rats, despite the decrease in RNA, but it was further increased by insulin treatment. Spermine content was decreased by diabetes, but was unchanged by insulin treatment. Thus the ratio spermidine/spermine in the adult diabetic rat was more typical of that seen in younger rats, whereas insulin treatment resulted in a ratio similar to that seen in rapidly growing tissues. 4. Ornithine decarboxylase activity was variable in the diabetic rat, showing a positive correlation with endogenous ornithine concentrations. This correlation was not seen in control or insulin-treated rats. Insulin caused a significant increase in ornithine decarboxylase activity relative to control or diabetic rats. 5. S-Adenosylmethionine decarboxylase activity was increased approx. 2-fold by diabetes and was not further affected by insulin. 6. Hepatic concentrations of the glucogenic amino acids, alanine, glutamine and glycine were decreased by diabetes. Their concentrations and that of glutamate were increased by injection of insulin. Concentrations of ornithine, proline, leucine, isoleucine and valine were increased in livers of diabetic rats and were decreased by insulin. Diabetes caused a decrease in hepatic concentration of serine, threonine, lysine and histidine. Insulin had no effect on serine, lysine and histidine, but caused a further fall in the concentration of threonine.     

Fetal rat islet insulin deficiency following maternal administration of streptozotocin

Pancreatic islets were isolated from the fetuses of normal rats and rats made diabetic by the iv administration of streptozotocin (STZ) on either Day 3 or 5 of pregnancy. Of the rats made diabetic on Day 3, one group also received insulin injections at the appearance of glucosuria. Maternal blood glucose on Day 20 of gestation was significantly different in the diabetic rats (405 +/- 27 mg/dl) from the normal (97 +/- 1 mg/dl) and insulin-treated diabetic rats (69 +/- 9 mg/dl). While fetal weight was significantly decreased in the STZ-treated rats (2.64 +/- 0.13 g vs 3.52 +/- 0.05 g for the control group, P less than 0.005), fetal glucose was significantly higher in the STZ-treated than in normal pups (342 +/- 11 vs 35 +/- 1 mg/dl, P less than 0.005). Both fetal weight and glucose were normalized by insulin treatment: 3.16 +/- 0.18 g and 31 +/- 7 mg/dl, respectively. Insulin release from fetal islets of diabetic dams was blunted after a week in culture both in basal and stimulated conditions. After 2 weeks in culture, there was partial recovery in the insulin response to glucose but it did not equal to that measured in fetal islets from the normal and insulin-treated diabetic rats. These data suggest maternal hyperglycemia severely impairs fetal weight and insulin release from fetal rat islets in vitro, and correction of the hyperglycemia by insulin treatment not only improves fetal weight and glucose concentrations, but it also normalizes insulin release from fetal rat islets in vitro.     

Diabetic nephropathy in KK and KK-Ay mice.

KK mice and KK-Ay mice were examined for age related changes in blood and urinary biophysiological parameters. Blood hemoglobin A1c levels were significantly higher in KK-Ay and KK mice as compared to non-diabetic ddY mice. In both diabetic mice, especially KK-Ay mice, plasma insulin levels markedly increased at 2 to 4 months of age, and the urinary glucose and microalbumin levels and albumin-to-creatinine ratios increased dependent on age. Plasma thrombomodulin levels significantly increased at 2 to 4 months of age in both KK and KK-Ay mice. Mild enlargement of mesangial matrix and segmental proliferative glomerular nephritis were revealed in KK and KK-Ay mice, respectively, at 4 months of age. KK-Ay mice with insulin resistance and high urine mAlb level might be useful as models for the early stage of diabetic nephropathy.     

A new mouse model of spontaneous diabetes derived from ddY strain.

By the selective breeding of obese male mice of the ddY strain and using indices of the heavy body weight and appearance of urinary glucose, we established two inbred strains in 1992: one with obesity and urinary glucose (Tsumura, Suzuki, Obese Diabetes: TSOD) and the other without them (Tsumura, Suzuki, Non Obesity: TSNO). The male TSOD mice constantly showed signs of obesity and urinary glucose with increases in food and water intake, body weight and some fat weight. The body mass index (BMI) clearly showed moderate obesity. Increases in the levels of diabetic blood parameters (glucose, insulin and lipids) were also found in males, in which the levels of blood glucose and insulin were high to the ages past the growth peak. In the histological studies, pancreatic islets of the TSOD males were found hypertrophic without any signs of insulitis or fibrous formation. Among these diabetic characteristics, some of which were similar to the reported models of non-insulin-dependent diabetes mellitus (NIDDM), the stable appearances of the hyperglycemia, the hyperinsulinemia and the hypertrophy of pancreatic islets to the ages past the growth peak were the prominent features. In these respect the TSOD mouse may be a useful model for researching the mechanisms of human diabetes and its complications.     

Diabetic crises in children treated with small doses of intramuscular insulin.

A new regimen for the management of diabetic crises in children using small doses of intramuscular insulin and generous amounts of intravenous fluids has been devised. Insulin dosage was calculated from the patient''s weight and was independent of the degree of hyperglycaemia. Minimum demands were made upon the laboratory.     

Effect of insulin pump therapy on blood pressure and the renin-angiotensin system of diabetic rats

Insulin therapy, administered by continuous subcutaneous infusion with osmotic pumps over a 28 day period at doses of 2.5 and 5.0 units/day, resulted in a statistically significant increase in body weight of diabetic rats. The concentration of blood glucose was reduced by 68% to 109 mg/dl blood sugar by the higher dose of insulin and only partial control of diabetes was achieved by the lower dose (185 mg/dl blood sugar, -39%). Blood pressure was normalized by both doses of insulin. Elevated serum angiotensin converting enzyme activity and plasma renin activity, expressed as generated angiotensin I, were unaffected by the lower dose of insulin, but were reduced by 26% and 40%, respectively at the higher dose. These data suggest that elevated serum ACE and plasma renin activity, commonly found in the streptozotocin-diabetic rat, may not be primarily responsible for hypertension in this model.     

Polyamines in pancreatic islets of obese-hyperglycemic (ob/ob) mice of different ages

To further evaluatethe role of polyamines in insulin production and cell replication indiabetic pancreatic islets, we have studied hyperplastic islets ofobese-hyperglycemic mice of different ages and normal islets of thesame strain. The aims of the study were to investigate the impact ofthe diabetic state and aging on polyamine contents and requirements inthese islets. Cultured islets from lean and obese animals containedsignificantly less polyamines than freshly isolated islets.Spermine-to-spermidine ratio was elevated in freshly isolated isletsfrom young obese mice compared with those from lean mice. In isletsfrom old obese animals, spermidine content was decreased, whereas thecontent of spermine was not different from that of young obese mice.The physiological significance of polyamines was investigated byexposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets butfailed to affect polyamine content of cell nuclei. Insulin content wasnot affected in polyamine-deficient islets of obese mice, irrespectiveof age, in contrast to decreased islet insulin content inpolyamine-depleted young lean animals. Polyamine depletion depressedDNA synthesis rate in obese mouse islets; in lean mice it actuallystimulated DNA synthesis. We concluded that important qualitative andquantitative differences exist between islets from obese-hyperglycemicand normal mice with respect to polyamine content and requirements ofpolyamines for regulation of insulin content and cell proliferation.The results suggest that spermine may be involved in mediating therapid islet cell proliferation noted early in obese-hyperglycemicsyndrome, but changes in spermine concentration do not seem to accountfor the decline in islet cell DNA synthesis in aged normoglycemic animals.

     

Reduced beta-cell mass and altered glucose sensing impair insulin-secretory function in betaIRKO mice

Pancreatic beta-cell-restricted knockout of the insulin receptor results in hyperglycemia due to impaired insulin secretion, suggesting that this cell is an important target of insulin action. The present studies were undertaken in beta-cell insulin receptor knockout (betaIRKO) mice to define the mechanisms underlying the defect in insulin secretion. On the basis of responses to intraperitoneal glucose, approximately 7-mo-old betaIRKO mice were either diabetic (25%) or normally glucose tolerant (75%). Total insulin content was profoundly reduced in pancreata of mutant mice compared with controls. Both groups also exhibited reduced beta-cell mass and islet number. However, insulin mRNA and protein were similar in islets of diabetic and normoglycemic betaIRKO mice compared with controls. Insulin secretion in response to insulin secretagogues from the isolated perfused pancreas was markedly reduced in the diabetic betaIRKOs and to a lesser degree in the nondiabetic betaIRKO group. Pancreatic islets of nondiabetic betaIRKO animals also exhibited defects in glyceraldehyde- and KCl-stimulated insulin release that were milder than in the diabetic animals. Gene expression analysis of islets revealed a modest reduction of GLUT2 and glucokinase gene expression in both the nondiabetic and diabetic mutants. Taken together, these data indicate that loss of functional receptors for insulin in beta-cells leads primarily to profound defects in postnatal beta-cell growth. In addition, altered glucose sensing may also contribute to defective insulin secretion in mutant animals that develop diabetes.     

Effect of maternal diabetes and ethanol interactions on embryo development in the mouse

The aim of this study was to determine the possible fetal effects of interaction between maternal diabetes and acute doses of alcohol. Pregnant TO mice were made diabetic by a single injection of streptozotocin (STZ) on gestation day (GD) 2. Single dose of 0.003 or 0.03 ml/g body weight of fresh ethanol (25% v/v of absolute alcohol in normal saline) was injected into groups of diabetic and nondiabetic animals on GD 7 or 8. One group of diabetic animals had a daily dose of 6-8 IU of insulin subcutaneously. Fetuses were collected on GD 18. There was a significant increase in the incidence of implantation failure in the diabetes plus ethanol groups and insulin control group. Ethanol injection on GD 7 accentuated diabetes-related embryonic resorption and intrauterine growth retardation (IUGR). This effect was less marked in the diabetic group treated with ethanol on GD 8. Diabetes alone produced a greater incidence of IUGR than ethanol alone. Midfacial hypoplasia and minor anomalies were found more frequently in the combination treatment groups. Holoprosencephaly and thymus hypoplasia observed in diabetic groups were found to be reduced in frequency in the diabetes plus ethanol groups, suggesting an antagonistic type of ethanol-diabetes interaction, stage-dependently. Since severely malformed embryos are known to be resorbed/killed in utero in mice, this reduction might reflect the magnitude of early death of severely malformed embryos. These data suggest that the interaction effects are possibly related to alterations in fundamental developmental processes of early embryos.     

Regional distribution of tyrosine,tryptophan, and their metabolites in the brain of epileptic El mice

Tyrosine, tryptophan, and their metabolites in the brain of ddY, non-stimulated El (El (–)), and stimulated El (El (+)) mice were measured using the three dimensional HPLC. The tryptophan content was lower in El (+) than ddY and El (–) mice. The 5-hydroxytrytophan content was much higher in both El groups. The serotonin content of El (+) was higher than that of ddY and El (–) mice. The kynurenine content was remarkably high in the El mice. The dopamine content was lower in El (–) than in ddY mice, whereas it was greater in El (+) than in El (–) mice. The norepinephrine showed higher levels in El (+) mice. These facts suggest that El mice posess congenital metabolic abnormalities of tryptophan and tyrosine and that kynurenine may play an important role as convulsant in El mice seizures along with changes in serotonin, dopamine, and norepinephrine that are inhibitory agents and responded to the repetitive convulsions.     

Potentiation of Insulin-Mediated Glucose Lowering without Elevated Hypoglycemia Risk by a Small Molecule Insulin Receptor Modulator

Insulin resistance is the key feature of type 2 diabetes and is manifested as attenuated insulin receptor (IR) signaling in response to same levels of insulin binding. Several small molecule IR activators have been identified and reported to exhibit insulin sensitization properties. One of these molecules, TLK19781 (Cmpd1), was investigated to examine its IR sensitizing action in vivo. Our data demonstrate that Cmpd1, at doses that produced minimal efficacy in the absence of insulin, potentiated insulin action during an OGTT in non-diabetic mice and enhanced insulin-mediated glucose lowering in diabetic mice. Interestingly, different from insulin alone, Cmpd1 combined with insulin showed enhanced efficacy and duration of action without increased hypoglycemia. To explore the mechanism underlying the apparent glucose dependent efficacy, tissue insulin signaling was compared in healthy and diabetic mice. Cmpd1 enhanced insulin’s effects on IR phosphorylation in both healthy and diabetic mice. In contrast, the compound potentiated insulin’s effects on Akt phosphorylation in diabetic but not in non-diabetic mice. These differential effects on signaling corresponding to glucose levels could be part of the mechanism for reduced hypoglycemia risk. The in vivo efficacy of Cmpd1 is specific and dependent on IR expression. Results from these studies support the idea of targeting IR for insulin sensitization, which carries low hypoglycemia risk by standalone treatment and could improve the effectiveness of insulin therapies.     

Insulin binding to circulating erythrocytes in nonobese diabetic mice

Insulin binding to circulating erythrocytes was studied in nonobese diabetic (NOD) mice which develop insulinopenic diabetes mellitus spontaneously. NOD mice with a short duration of diabetes mellitus and mild insulinopenia did not show any change in insulin binding, while those with a long duration of diabetes mellitus and severe insulinopenia showed an increase in insulin binding compared with nondiabetic NOD mice (6.85 +/- 0.38% bound vs. 4.19 +/- 0.24% bound, p less than 0.01). This increase in insulin binding was due to an increase in the number of receptors. Insulin treatment of diabetic NOD mice significantly reduced the insulin binding by 64%, which resulted from a decrease in the number of the receptors. These results indicate that insulin binding to erythrocytes in NOD mice is controlled mostly by up-and-down regulation.     

Diabetic state-induced modifications of succinylcholine binding mode in the microsomal fractions of mouse skeletal muscles

The skeletal muscles of alloxan-induced diabetic mice and genetically diabetic KK-CAY mice are hypersensitive to a depolarizing blocker, succinylcholine (SuCh) but not to the competitive antagonist, d-tubocurarine (d-TC). The mechanism by which the action of the depolarizing blocker is modified in the diabetic state was investigated on the binding of 14C-SuCh to the microsomal fraction isolated from mouse skeletal muscles. The Scatchard plot of microsomal preparations from normal ddY mice showed positive cooperativity in SuCh binding, whereas that of the preparations from alloxan-induced diabetic mice as well as genetically diabetic KK-CAY mice lost the positive cooperative interactions. The dissociation constant (Kd) of high affinity site in diabetic muscles was significantly lower than that in non-diabetic ddY muscle. The microsomal fractions from denervated muscles of normal ddY mice maintained weakly positive cooperativity in SuCh binding, and the affinity of SuCh binding in denervated muscles was lower than that of non-denervated muscles. In conclusion, the diabetic state selectively altered the SuCh binding mode. This alteration seems to be closely correlated with the pharmacological hypersensitivity to SuCh.     

Deficient production of the lymphokine eosinophil stimulation promoter in chemically induced and mutation diabetes mellitus in mice.

The cellular defects possibly responsible for diminished in vivo granuloma formation in diabetic Schistosoma mansoni-infected mice were investigated. Diabetic and control animals develop a similar degree of eosinophilia. Eosinophils obtained from diabetic mice also respond normally to the lymphokine eosinophil stimulation promoter (ESP). Lymphoid cells of chemically induced (streptozotocin) and mutation diabetic (db/db) mice, however, have a decreased capacity to produce/secrete ESP in response to soluble egg antigens of S. mansoni. Administration of insulin to diabetic mice is associated with a partial reversal of the decreased ability of their lymphoid cells to generate ESP. These findings show that defective cellular immunity in diabetic animals may be partially explained by the failure of their lymphocytes to produce the soluble mediators involved in recruitment of target cells.     

Investigation of central mechanism of insulin induced hypoglycemic convulsions in mice

Insulin produces seizures in healthy and diabetic animals. Amongst suggested mechanisms, the role of neuromodulators and neurotransmitters is not clear. The present study explores the mechanisms involved in insulin-induced convulsions. Convulsions were induced in Swiss male albino mice with graded doses of insulin. Blood sugar levels were measured prior to and after the first convulsion. Drugs like 5-HTP (5-HT precursor), pCPA (5-HT depletor), ondansetron (5-HT3 antagonist), ketanserin (5-HT, antagonist), ketamine (NMDA antagonist), 1-dopa (dopamine precursor) and reserpine (amine depletor) were studied for interaction with convulsive behaviour induced by insulin. Insulin in 2 IU/kg dose did not produce convulsions while 4 and 8 IU/kg doses produced convulsions in 50% and 100% of animals respectively. 5-HTP, ondansetron, ketanserin, ketamine and l-dopa significantly protected/inhibited animals from convulsions at all studied doses of insulin. On the contrary, pCPA and reserpine potentiated insulin induced convulsions. Insulin caused mortality in 40 and 100% animals with 4 and 8 IU/kg doses respectively. pCPA and reserpine treatments caused mortality at all doses of insulin, while other drugs did not influence insulin induced mortality. Blood sugar levels were reduced in all groups irrespective of the presence or absence of convulsions. A definitive link of serotonergic, dopaminergic and excitatory amino acid pathways in mediating insulin-induced hypoglycemic convulsions is suggested.     

Effects of Se-enriched polysaccharides produced by Enterobacter cloacae Z0206 on alloxan-induced diabetic mice

In this study, the water-soluble selenium-enriched exopolysaccharides (Se-ECZ-EPS) were isolated from submerged culture broth of Enterobacter cloacae Z0206 through fermentation, ethanol precipitation and deproteinization. The protective effects of Se-ECZ-EPS on alloxan-induced diabetic mice were investigated. Diabetes was induced in ICR (Institute of Cancer Research) mice by administration of single doses of alloxan intraperitoneally (190 mg/kg body weight). Se-ECZ-EPS at a dose of 200 mg/kg body weight were administered per os (p.o.) as single dose per day to diabetes-induced mice for a period of 42 days. The decrease in body weight, serum insulin level, and the increase in blood glucose level, glycosylated serum protein (GSP), total cholesterol (TC) and triglycerides (TG) in liver were observed in diabetic mice. On the other hand, oral administration of Se-ECZ-EPS resulted in a significant reduction in fasting blood glucose levels, GSP, TC and TG contents in liver coupled with improvement of body weight and serum insulin level in comparison with diabetic control group. These results suggest that Se-ECZ-EPS possess significant protective and anti-diabetic effects in alloxan-induced diabetic mice.