Agouti-related protein is a mediator of diabetic hyperphagia

To explore the role of agouti-related protein (AGRP) in diabetic hyperphagia changes in hypothalamic AGRP mRNA levels were examined in diabetic rats. Rats rendered diabetic by streptozotocin displayed marked hyperglycemia (blood glucose 456.0+/-8.4 mg/dl versus 71.8+/-1.9 mg/dl) and hyperphagia (36.9+/-1.0 g/day versus 22.0+/-0.4 g/day), that was associated with a 286.6+/-4.4% increase in hypothalamic AGRP mRNA and a 178.9+/-13.5% increase in hypothalamic NPY mRNA. Insulin treatment of diabetic rats partially corrected blood glucose (147.4+/-13.1 mg/dl) and ameliorated hyperphagia (26.6+/-2.0 g/day). Insulin replacement was also associated with a return of hypothalamic AGRP mRNA (111.7+/-8.3% of controls) and NPY mRNA (125.0+/-8.9% of controls) from the elevated levels that were observed in untreated diabetic rats. In contrast to insulin treated rats, sodium orthovanadate treated diabetic rats remained significantly hyperglycemic (361.5+/-12.5 mg/dl). However, despite their persistent hyperglycemia, orthovanadate treated diabetic rats were still observed to have a significant reduction of hypothalamic AGRP mRNA (138.7+/-11.4%) and NPY mRNA (129.9+/-9.8%). Simultaneous measurement of serum leptin revealed suppressed levels in both untreated diabetic (0.5+/-0.1 ng/ml) and sodium orthovanadate treated rats (0.5+/-0.1 ng/ml) compared to non-diabetic controls (2.1+/-0.1 ng/ml). These data indicate that AGRP is a mediator of diabetic hyperhpagia and suggest that insulin can directly influence hypothalamic AGRP and NPY mRNA expression.     

Miglitol (BAY m 1099) Treatment of Diabetic Hypothalamic-Dietary Obese Rats Improves Islet Response to Glucose

AXEN, KATHLEEN V., XUE LI, AND ANTHONY SCLAFANI. Miglitol (BAY m 1099) treatment of diabetic hypothalamic-dietary obese rats improves islet response to glucose. Obes Res. 1999;7:83–89. Objective : The well-absorbed α-glucosidase inhibitor, miglitol (BAY m 1099), was included in the diets of hypothalamic-dietary obese diabetic rats to investigate its ability to improve glycemia and thereby reverse glucotoxic effects on islet secretory response. Research Methods and Procedures : Female rats received bilateral electrolytic lesions of the ventromedial hypothalamus and were fed high-fat, sucrosesupplemented diets until hyperinsulinemia and hyperglycemia were observed after 3 hours of food deprivation (nonfed). Diabetic animals were assigned to miglitol-treated (40 mg/17 g of diet) or untreated groups for 3 weeks; pancreatic islets were isolated for incubation experiments. Results : No differences in food intake, body weights, or nonfed plasma glucose or insulin levels were seen between treated and untreated diabetic rats. Islets isolated from untreated diabetic rats showed elevated basal insulin release and no insulin secretory response to an elevation in glucose concentration. In contrast, islets obtained from miglitol-treated rats showed more normal basal release and a significant insulin secretory response to glucose. Incubation of islets, obtained from normal control rats or untreated diabetic rats, in media containing miglitol at levels estimated to exist in plasma of treated rats had no effect on islet insulin secretory responses to glucose. Discussion : Islet secretory response was improved despite continued hyperglycemia and severe insulin resistance. Miglitol treatment may improve islet sensitivity to glucose either through effects on islet metabolism requiring prolonged exposure or by improvement in postmeal glycemia, despite persistent hyperglycemia.     

Glucokinase in B-cell-depleted islets of Langerhans

Glucose phosphorylation was studied in B-cell-enriched or in B-cell-depleted pancreatic islets from normal or streptozotocin-diabetic rats, respectively, using quantitative histochemical procedures. The data indicate that B-cell-enriched preparations from normal animals and whole islets from normals, diabetics, and insulin-treated diabetic animals have comparable glucokinase activities. Average maximum velocities were (mmol/kg dry tissue/hr) 134.1 +/- 7.3 for whole islets and 125.6 +/- 10.7 for the B-cell-enriched preparations from normal rats, 143.1 +/- 13.6 for B-cell-depleted islets from diabetic rats, and 124.4 +/- 10.7 for B-cell-depleted islets from insulin-treated diabetic animals. The Kmax for glucose of the enzyme in islets from untreated diabetic rats was 16 mM, comparable to the Kmax found for glucokinase from normal rat islets. Mannoheptulose, previously shown to be a competitive inhibitor of glucokinase from liver and normal islets, also inhibited glucokinase in B-cell-depleted islets from diabetic rats. The data indicate that glucokinase is not selectively located in the B-cell, as was previously assumed, but is also found in A- and/or D-cells of diabetic rats. This observation raises significant questions about the functional role of islet glucokinase under control and diabetic conditions.     

Characterization of STZ-Induced Type 2 Diabetes in Zucker Fatty Rats

The Zucker fatty (ZF) rat is a disease model of obesity and metabolic syndrome, such as hyperlipidemia and insulin resistance, resulting from hyperphagia owing to the loss of function of the leptin receptor, but it rarely develops hyperglycemia. We examined the effects of different doses of streptozotocin (STZ). A low dosage of STZ (30 mg/kg body weight, i.p.) elevated blood glucose levels in ZF rats up to 300 mg/dl within a week, and to nearly 500 mg/dl by 5 weeks after injection of STZ. Besides hyperglycemia, STZ-treated ZF (STZ-ZF) rats retained metabolic syndrome features such as hyperlipidemia and hyperinsulinemia. The stimulated insulin secretion in response to orally-loaded glucose disappeared completely in STZ-ZF rats. Although there were no significant differences in the morphology of pancreatic islets between vehicle-treated ZF (Cont-ZF) and STZ-ZF rats, the insulin content was markedly decreased in STZ-ZF rats. The hepatic gene expression for gluconeogenic enzymes was upregulated in STZ-ZF rats compared with Cont-ZF rats. Metformin lowered the blood glucose levels of STZ-ZF rats in a dose-dependent manner. These results suggest that STZ-ZF rats are useful for studies of T2DM and for the evaluation of the efficacy of anti-diabetic drugs.     

Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death

Hepatocyte growth factor (HGF) increases beta cell proliferation and function in rat insulin promoter (RIP)-targeted transgenic mice. RIP-HGF mouse islets also function superiorly to normal islets in a transplant setting. Here, we aimed to determine whether viral gene transfer of the HGF gene into mouse islets ex vivo could enhance the performance of normal islets in a streptozotocin-diabetic severe combined immunodeficient mouse marginal islet mass model in which 300 uninfected or adenovirus (Adv) LacZ-transduced islet equivalents were insufficient to correct hyperglycemia. In dramatic contrast, 300 AdvHGF-transduced islet equivalents promptly (day 1) and significantly (p < 0.01) decreased random non-fasting blood glucose levels, from 351 +/- 20 mg/dl to an average of 191 +/- 7 mg/dl over 8 weeks. At day 1 post-transplant, beta cell death was significantly (p < 0.05) decreased, and the total insulin content was significantly (p < 0.05) increased in AdvHGF-transduced islets containing grafts. This anti-beta cell death action of HGF was independently confirmed in RIP-HGF mice and in INS-1 cells, both treated with streptozotocin. Activation of the phosphatidylinositol 3-kinase/Akt intracellular-signaling pathway appeared to be involved in this beta cell protective effect of HGF in vitro. In summary, adenoviral delivery of HGF to murine islets ex vivo improves islet transplant survival and blood glucose control in a subcapsular renal graft model in immuno-incompetent diabetic mice.     

Characterization of streptozotocin-induced diabetic rats and pharmacodynamics of insulin formulations

Morphological and functional changes of rat pancreatic islets caused by administration of streptozotocin (STZ) and the bioavailability of insulin formulations administered to STZ-induced diabetic rats with fasting (12 h) or non-fasting were investigated. Islets isolated from normal rats maintained a good three-dimensional structure and the islet yield was 962.5+/-86.5 islet equivalent number (IEQ, islets converted to an average diameter of 150 microm). In the diabetic group (>500 mg/ml blood glucose), the islet yield was only 44.4+/-8.3 IEQ and the islet was severely damaged. The minimum reduction of blood glucose of each formulation, such as insulin solution, microcrystal, and insulin microcrystal capsule, was shown to be 11.3, 11.0, and 16.3 mg/dl, respectively, at 6 h in fasting with diabetic rats. These results indicated that the administration of insulin formulations to the fasting groups increased the severe hypoglycemic effect of insulin action more than in non-fasting diabetic rats. The diabetic rat with fasting has a regulatory disorder in maintaining the blood glucose level. Accordingly, the validity of pharmacological availability as an optimal modeling of insulin formulations is best in non-fasting STZ-induced diabetic rats.     

Subsensitivity to insulin in adipocytes from rats submitted to foot-shock stress

We examined the effect of three daily foot-shock stress sessions on glucose homeostasis, insulin secretion by isolated pancreatic islets, insulin sensitivity of white adipocytes, and glycogen stores in the liver and soleus muscle of rats. Stressed rats had plasma glucose (128.3 +/- 22.9 mg/dL) and insulin (1.09 +/- 0.33 ng/mL) levels higher than the controls (glucose, 73.8 +/- 3.5 mg/dL; insulin, 0.53 +/- 0.11 ng/mL, ANOVA plus Fisher's test; p < 0.05). After a glucose overload, the plasma glucose, but not insulin, levels remained higher (area under the curve 8.19 +/- 1.03 vs. 4.84 +/- 1.33 g/dL 30 min and 102.7 +/- 12.2 vs. 93.2 +/- 16.1 ng/mL 30 min, respectively). Although, the area under the insulin curve was higher in stressed (72.8 +/- 9.8 ng/mL) rats than in control rats (34.9 +/- 6.9 ng/mL) in the initial 10 min after glucose overload. The insulin release stimulated by glucose in pancreatic islets was not modified after stress. Adipocytes basal lipolysis was higher (stressed, 1.03 +/- 0.14; control, 0.69 +/- 0.11 micromol of glycerol in 60 min/100 mg of total lipids) but maximal lipolysis stimulated by norepinephrine was not different (stressed, 1.82 +/- 0.35; control, 1.46 +/- 0.09 micromol of glycerol in 60 min/100 mg of total lipids) after stress. Insulin dose-dependently inhibited the lipolytic response to norepinephrine by up to 35% in adipocytes from control rats but had no effect on adipocytes from stressed rats. The liver glycogen content was unaltered by stress, but was lower in soleus muscle from stressed rats than in control rats (0.45 +/- 0.04 vs. 0.35 +/- 0.04 mg/100 mg of wet tissue). These results suggest that rats submitted to foot-shock stress develop hyperglycemia along with hyperinsulinemia as a consequence of insulin subsensitivity in adipose tissue, with no alteration in the pancreatic sensitivity to glucose. Foot-shock stress may therefore provide a useful short-term model of insulin subsensitivity.     

Changes in insulin response to glucose after exercise training in partially pancreatectomized rats

The effects of exercise training on glucose-stimulated insulin secretion (GSIS) were studied in male Sprague-Dawley rats made mildly to severely diabetic by partial pancreatectomy. Exercise trained (10 wk treadmill; T) and untrained (Unt) rats were grouped according to posttraining fed-state hyperglycemia as follows: T less than 200 and Unt less than 200 (glucose concn less than 200 mg/dl), T 200-300 and Unt 200-300 (glucose concn 200-300 mg/dl), and T greater than 300 and Unt greater than 300 (glucose concn greater than 300 mg/dl). After exercise training, hyperglycemic glucose clamps were performed in awake rats by elevation of arterial blood glucose concentration 126 mg/dl above fasting basal levels for 90 min. Exercise training significantly increased muscle citrate synthase activity. Prevailing hyperglycemia was reduced during the 10-wk exercise training period in all T rats with fed-state glucose concentrations less than 300, and only 53% of Unt rats in these groups had reduced glycemia. GSIS was significantly higher in T less than 200 [2.4 +/- 0.7 (SD) ng/ml at 90 min] than in Unt less than 200 (1.5 +/- 0.3). A similar response was found for T 200-300 (1.1 +/- 0.3 ng/dl) vs. Unt 200-300 (0.7 +/- 0.1) but not T greater than 300 (0.36 +/- 0.2) vs Unt greater than 300 (0.44 +/- 0.05). Sham-operated control rats had insulin concentrations of 6.6 +/- 1.6 ng/ml at the 90th min of the clamp. Acute exercise reduced fed-state glycemia in rats with mild-to-moderate (less than 300 mg/dl) diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ginseng berry reduces blood glucose and body weight in db/db mice.

In this study, we observed anti-diabetic and anti-obesity effects of Panax ginseng berry in adult C57BL/Ks db/db mice and their lean littermates. Animals received daily intraperitoneal injections of Panax ginseng berry extract at 150 mg/kg body wt. for 12 consecutive days. On Day 5, the extract-treated db/db mice had significantly lower fasting blood glucose levels as compared to vehicle-treated mice (180.5+/-10.2 mg/dl vs. 226.0+/-15.3 mg/dl, P < 0.01). On day 12, the extract-treated db/db mice were normoglycemic (134.3+/-7.3 mg/dl) as compared to vehicle-treated mice (254.8+/-24.1 mg/dl; P < 0.01). Fasting blood glucose levels of lean mice did not decrease significantly after treatment with extract. After 12 days of treatment with the extract, glucose tolerance increased significantly, and overall blood glucose exposure calculated as area under the curve (AUC) decreased 53.4% (P < 0.01) in db/db mice. Furthermore, db/db mice treated with extract (150 mg/kg body wt.) showed weight loss from 51.0+/-1.9 g on Day 0, to 46.6+/-1.7 g on Day 5, and to 45.2+/-1.4 g on Day 12 (P < 0.05 and P < 0.01 compared to Day 0, respectively). The body weight of lean littermates also decreased at the same dose of extract. These data suggest that Panax ginseng berry extract may have therapeutic value in treating diabetic and obese patients.     

Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: mechanisms and implications for preclinical models

The effects of anesthetic agents, commonly used in animal models, on blood glucose levels in fed and fasted rats were investigated. In fed Sprague-Dawley rats, ketamine (100 mg/kg)/xylazine (10 mg/kg) (KX) produced acute hyperglycemia (blood glucose 178.4 +/- 8.0 mg/dl) within 20 min. The baseline blood glucose levels (104.8 +/- 5.7 mg/dl) reached maximum levels (291.7 +/- 23.8 mg/dl) at 120 min. Ketamine alone did not elevate glucose levels in fed rats. Isoflurane also produced acute hyperglycemia similar to KX. Administration of pentobarbital sodium did not produce hyperglycemia in fed rats. In contrast, none of these anesthetic agents produced hyperglycemia in fasted rats. The acute hyperglycemic effect of KX in fed rats was associated with decreased plasma levels of insulin, adrenocorticotropic hormone (ACTH), and corticosterone and increased levels of glucagon and growth hormone (GH). The acute hyperglycemic response to KX was dose-dependently inhibited by the specific alpha2-adrenergic receptor antagonist yohimbine (1-4 mg/kg). KX-induced changes of glucoregulatory hormone levels such as insulin, GH, ACTH, and corticosterone were significantly altered by yohimbine, whereas the glucagon levels remained unaffected. In conclusion, the present study indicates that both KX and isoflurane produce acute hyperglycemia in fed rats. The effect of KX is mediated by modulation of the glucoregulatory hormones through stimulation of alpha2-adrenergic receptors. Pentobarbital sodium did not produce hyperglycemia in either fed or fasted rats. Based on these findings, it is suggested that caution needs to be taken when selecting anesthetic agents, and fed or fasted state of animals in studies of diabetic disease or other models where glucose and/or glucoregulatory hormone levels may influence outcome and thus interpretation. However, fed animals are of value when exploring the hyperglycemic response to anesthetic agents.     

Hypoglycemic and hypolipidemic effects of aqueous extract of Arachis hypogaea in normal and alloxan-induced diabetic rats.

The hypoglycemic and hypolipidemic effect of aqueous extract of Arachis hypogaea was investigated in normal and alloxan-induced diabetic rats. The extract caused a significant (P < 0.05) decrease of fasting blood glucose of both normal and alloxan-induced diabetic rats from 102.60 +/- 1.65 mg/dl to 88.79 +/- 0.94 mg/dl for normal and 189.0 +/- 30.79 mg/dl to 107.55 +/- 1.54 mg/dl for alloxan-induced diabetic rats. The extract also caused a significant (P < 0.05) decrease in serum triglyceride, total cholesterol, HDL-cholesterol and LDL-cholesterol in both normal and alloxan-induced diabetic rats.     

Hyperglycemia does not increase basal hypothalamo-pituitary-adrenal activity in diabetes but it does impair the HPA response to insulin-induced hypoglycemia

Recently, we established that hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia were impaired in uncontrolled streptozotocin (STZ)-diabetic (65 mg/kg) rats and insulin treatment restored most of these responses. In the current study, we used phloridzin to determine whether the restoration of blood glucose alone was sufficient to normalize HPA function in diabetes. Normal, diabetic, insulin-treated, and phloridzin-treated diabetic rats were either killed after 8 days or subjected to a hypoglycemic (40 mg/dl) glucose clamp. Basal: Elevated basal ACTH and corticosterone in STZ rats were normalized with insulin but not phloridzin. Increases in hypothalamic corticotrophin-releasing hormone (CRH) and inhibitory hippocampal mineralocorticoid receptor (MR) mRNA with STZ diabetes were not restored with either insulin or phloridzin treatments. Hypoglycemia: In response to hypoglycemia, rises in plasma ACTH and corticosterone were significantly lower in diabetic rats compared with controls. Insulin and phloridzin restored both ACTH and corticosterone responses in diabetic animals. Hypothalamic CRH mRNA and pituitary pro-opiomelanocortin mRNA expression increased following 2 h of hypoglycemia in normal, insulin-treated, and phloridzin-treated diabetic rats but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal MR mRNA in control, insulin-, and phloridzin-treated diabetic rats but not uncontrolled diabetic rats, whereas glucocorticoid receptor mRNA was not altered by hypoglycemia. In conclusion, despite elevated basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that defects in the CRH response may be related to a defective MR response. It is intriguing that phloridzin did not restore basal HPA activity but it restored the HPA response to hypoglycemia, suggesting that defects in basal HPA function in diabetes are due to insulin deficiency, but impaired responsiveness to hypoglycemia appears to stem from chronic hyperglycemia.     

Hormone secretion and glucose metabolism in islets of Langerhans of the isolated perfused pancreas from normal and streptozotocin diabetic rats.

The glucose responsiveness of alpha- and beta-cells of normal as well as untreated and insulin-treated streptozotocin diabetic rats was tested in the extracorporeal perfusion system. Also assessed was the possible in vitro effect of added insulin on the glucose sensitivity of islets from untreated diabetic animals. Insulin and glucose responsiveness of the two cell types. The rate of glucose entry islet tissue was estimated, and the effect of glucose on the tissue supply of ATP and lactate and the cyclic 3':5'-AMP level of islets was measured under the above in vitro conditions. It was demonstrated that beta-cells are more accessible to glucose than alpha-cells, that glucose entry into islet cells is not significantly modified by insulin and that glucose had no effect on ATP, lactate and cyclic 3':5'-AMP levels of islet tissue under any of the conditions investigated. High insulin in vitro elevated ATP levels of alpha-cell islets independent of extracellular glucose. Glucose caused insulin release from normal but not from diabetic islets and rapidly and efficiently suppressed stimulated glucagon secretion of the pancreas from normal and insulin treated diabetic rats. Glucose was less effective in inhibiting stimulated glucagon secretion by the pancreas from untreated diabetic rats whether insulin was added to the perfusion media or not. Therefore, profound differences of glucose responsiveness of alpha-cells fail to manifest themselves in alterations of basic parameters of glucose and energy metabolism in contrast to what had been postulated in the literature. It is however, apparent that the glucose responsiveness of alpha-cells is modified by insuling by an as yet undefined mechanism.     

Effects of Insulin on Altered Mechanical and Electrical Papillary Muscle Activities of Diabetic Rats

Since insulin compounds can restore some metabolic parameters and lipid profile alterations of the diabetic rat heart, we investigated whether these beneficial effects extend to diabetic rat cardiac dysfunctions. Twenty-four male Wistar albino rats, 6 months of age with an average body weight of 250–320 g, were divided randomly into three groups, each consisting of eight rats: control-group (C) rats were fed with standard rat nutrient and water; diabetic-group (D) rats were treated with a single intramuscular injection of streptozotocin (STZ, 45 mg/kg), dissolved in 0.01 M sodium citrate, pH adjusted to 4.5; and insulin-treated diabetic group (D + INS) rats were treated with subcutaneous injections of 1 IU/l insulin (INS) twice a day after a single intramuscular injection of STZ (45 mg/kg). Treatment of D rats with INS caused a time-dependent decrease in blood glucose. We found that the lipid profile and HbA_1c levels in the D + INS group reached the values of control rats at the end of the treatment period. Contraction force in group D was compared with values from groups C and D + INS (p < 0.05). Values were obtained at a muscle contraction and relaxation time of milliseconds, with contraction time in D compared to C and D compared to D + INS and C (p < 0.05). Rate-dependent changes in action potential configuration in left ventricular papillary muscle obtained from 8-week control, STZ-treated D and D + INS rats showed significant membrane potential changes between C and STZ-treated D animals. Action potential amplitude showed significant changes between matched D + INS and STZ-treated D animals. Depolarization time showed significant changes between C and STZ-treated D animals and between the D + INS and D groups. Half-repolarization time showed significant changes between D + INS and STZ-treated D animals and compared to the D and C groups. Our data suggest that the beneficial effects of insulin treatment on the mechanical and electrical activities of the diabetic rat heart appear to be due to restoration of the diminished K⁺ currents, partially related to the restoration of hyperglycemia.     

Anti-hyperglycemic effect of the polysaccharides fraction from American ginseng berry extract in ob/ob mice.

In this study, we evaluated the anti-hyperglycemic effect of a polysaccharides fraction from American ginseng berry extract in diabetic ob/ob mice. All animals received daily intraperitoneal injections of polysaccharides at 150 mg/kg body wt. (n = 5), polysaccharides at 50 mg/kg body wt. (n = 5), or vehicle (n = 5) for 10 consecutive days. On Day 5, as compared to the vehicle-treated mice (230.5 +/- 13.5 mg/dl, mean +/- S.E), mice from both treated groups showed significantly lower fasting blood glucose levels (187.4 +/- 20.5 mg/dl and 187.4 +/- 17.1 mg/dl), respectively (both P < 0.05). On Day 10, compared to the vehicle group (240.1 +/- 12.3 mg/dl), the 50 mg/kg dose group were at 188.4 +/- 12.6 mg/dl (P < 0.05), and the 150 mg/kg dose group were normoglycemic (148.8 +/- 17.6 mg/dl, P < 0.01). Those ob/ob mice treated with vehicle did not, however, show significant changes in fasting blood glucose levels. Data from the intraperitoneal glucose tolerance test (IPGTT) showed that, compared to Day 0, there was a significant improvement in glucose tolerance in animals who received the 50 and 150 mg/kg polysaccharide doses, and the area under the curve (AUC) decreased 15.5% (P < 0.05) and 28.2% (P < 0.01), respectively. Interestingly, after cessation of polysaccharide treatment, the fasting blood glucose levels stayed lower, and returned to control concentration on Day 30. We also observed that the polysaccharides fraction did not affect body weight changes in ob/ob mice. Our data suggest that the polysaccharides fraction from American ginseng berry extract has a potential clinical utility in treating diabetic patients.     

Characterization of Streptozotocin-induced Diabetic Rats and Pharmacodynamics of Insulin Formulations

Morphological and functional changes of rat pancreatic islets caused by administration of streptozotocin (STZ) and the bioavailability of insulin formulations administered to STZ-induced diabetic rats with fasting (12 h) or non-fasting were investigated. Islets isolated from normal rats maintained a good three-dimensional structure and the islet yield was 962.5±86.5 islet equivalent number (IEQ, islets converted to an average diameter of 150 μm). In the diabetic group (>500 mg/ml blood glucose), the islet yield was only 44.4±8.3 IEQ and the islet was severely damaged. The minimum reduction of blood glucose of each formulation, such as insulin solution, microcrystal, and insulin microcrystal capsule, was shown to be 11.3, 11.0, and 16.3 mg/dl, respectively, at 6 h in fasting with diabetic rats. These results indicated that the administration of insulin formulations to the fasting groups increased the severe hypoglycemic effect of insulin action more than in non-fasting diabetic rats. The diabetic rat with fasting has a regulatory disorder in maintaining the blood glucose level. Accordingly, the validity of pharmacological availability as an optimal modeling of insulin formulations is best in non-fasting STZ-induced diabetic rats.     

Feeble bronchomotor responses in diabetic rats in association with decreased sensory neuropeptide release

Type I diabetes is associated with a low incidence of asthma. We tested whether a decrease in sensory neuropeptide release is associated with an attenuated bronchoconstrictive response to field stimulation (FS; 100 stimuli, 20 V, 0.1 ms, 20 Hz) in streptozotocin (STZ)-induced diabetes. The organ fluid of the preparations were also tested for substance P, calcitonin gene-related peptide (CGRP), and somatostatin concentrations by RIA. Preparations were from either normal rats or those pretreated with 50 mg/kg STZ iv 8 wk before experiment. A group of STZ-treated animals was supplied with insulin delivery (4 IU/day sc) implants between 4 and 8 wk. A subgroup was formed to study the effect of capsaicin desensitization. The atropine-resistant contraction was attenuated by diabetes without capsaicin-sensitive relaxation response. Exogenous CGRP and substance P potentiated, whereas somatostatin inhibited (1 nM-10 microM) the FS-induced contractions in rings from either group. FS released somatostatin, CGRP, and substance P from 0.17 +/- 0.024, 0.15 +/- 0.022, and 1.65 +/- 0.093 to 0.58 +/- 0.032, 0.74 +/- 0.122, and 5.34 +/- 0.295 in preparations from normal, and from 0.19 +/- 0.016, 0.11 +/- 0.019, and 0.98 +/- 0.116 to 0.22 +/- 0.076, 0.34 +/- 0.099, and 1.84 +/- 0.316 fmol/mg wet wt in preparations from diabetic rats. Insulin supplementation restored neuropeptide release in rings from STZ-treated rats. The results show that the decreased FS-induced contractions occurred with a decrease in sensory neuropeptide release in STZ-diabetic rats.     

Insulin and glucagon in rats with islet cell tumors induced by small doses of streptozotocin

Plasma insulin and glucagon responses to oral glucose loading were examined in rats with islet cell tumors induced by a single intravenous injection of streptozotocin (30 or 40 mg/kg body weight). Twenty-four macroscopic and six microscopic tumors occurred in 21 rats. In 15 of 21 tumor-bearing rats, there was exaggerated insulin release in response to oral glucose. Plasma glucose levels did not rise with the oral glucose load and were comparable to those seen in normal animals. Hence these rats are described as having "responsive tumors." In six rats with "nonresponsive tumors" there was no insulin response and the plasma glucose levels rose. No significant differences in plasma levels were observed between the two groups. Nonresponsive tumors as well as responsive tumors contained a significant amount of extractable insulin (17.68 +/- 8.60 and 35.07 +/- 10.05 mg/g wet weight, respectively) and detectable amounts of immunoreactive glucagon (1.47 +/- 0.61 and 2.24 +/- 0.67 micrograms/g wet weight, respectively). These results suggest that a small dose of streptozotocin produces two types of islet cell tumors. One is insulin producing and insulin secreting whereas the other is insulin producing but not insulin secreting.     

Possible effect of bromocriptine on the insulin receptor

The administration of 0.00011 mg/g weight/day of bromocriptine (CB154) for 7 days to Wistar rats, improved the peripheral glucose uptake without significant changes in plasma insulin level, during the intravenous glucose tolerance test (0.33 g/kg). The mode of the bromocriptine action on binding of 125I insulin to erythrocyte insulin receptors has been evaluated. The total number of sites was greater with bromocriptine (513.1 +/- 124.1 pM/1,CB154 815.6 +/- 107.9 pM/l) (p less than 0.01). The high affinity/low capacity compound of insulin receptor, in CB154 rats (51.8 +/- 16.8 pM/l) was higher than in normal rats (18.3 +/- 8.9 pM/l) (p less than 0.005). Additional studies indicated that CB154 had no effect on the rate of association and dissociation of 125I insulin from rats erythrocyte insulin receptors. The degradation of insulin or the erythrocyte receptor sites do not change, after the treatment with CB154.     

Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle

The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.