The beta cell function in NIDDM patients with secondary failure: a three year follow-up of combined oral hypoglycemic and insulin therapy.

Eleven Type 2 (non-insulin-dependent) diabetic patients, islet cell autoantibodies negative, nonobese with secondary failure to oral hypoglycemic agents (OHA) [glyburide (7.5 mg/day) and phenformin (75 mg/day)] and HbA1c 10.2 +/- 0.6% were studied. Insulin receptors on circulating monocytes, glucose utilization at supraphysiological insulin concentrations, and plasma C-peptide after i.v. glucagon were evaluated before and after 2 months of combined therapy with OHA and insulin (Ultratard HM Novo). A significant improvement was demonstrated in HbA1c and glycemia after two months of treatment. Glucose MCR was increased after two months of treatment whilst basal C-peptide was decreased as well as receptor binding to monocytes. After three years of combined therapy, body weight, glycemia and HbA1c did not increase. After three years the C-peptide basal values were significantly increased with respect to values found after 2 months of therapy. These results demonstrate that insulin treatment may restore insulin sensitivity in NIDDM patients resistant to OHA treatment and that after three years there is no exhaustion of B-cell function.     

Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

OBJECTIVE--To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. DESIGN--Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. SETTING--Study in normal people at a diabetes research unit and a university department of medical physics. SUBJECTS--Seven healthy male volunteers aged 20-39 not receiving any other drugs. INTERVENTIONS--After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. END POINT--To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and more dramatic hypoglycaemic effect with the insulin analogue. CONCLUSIONS--The much faster absorption from subcutaneous tissue of the disubstituted monomeric insulin analogue compared with soluble insulin suggests that the analogue may be a potential candidate for rapid insulin delivery after subcutaneous bolus injection.     

Central action of insulin regulates pulsatile luteinizing hormone secretion in the diabetic sheep model

This study tested the hypothesis that central mechanisms regulating luteinizing hormone (LH) secretion are responsive to insulin. Our approach was to infuse insulin into the lateral ventricle of six streptozotocin-induced diabetic sheep in an amount that is normally present in the CSF when LH secretion is maintained by peripheral insulin administration. In the first experiment, we monitored cerebrospinal fluid (CSF) insulin concentrations every 3-5 h in four diabetic sheep given insulin by peripheral injection (30 IU). The insulin concentration in the CSF was increased after insulin injection, and there was a positive relationship between CSF and plasma concentrations of insulin (r = 0.80, P < 0.01). In the second experiment, peripheral insulin administration was discontinued, and the sheep received either an intracerebroventricular (i.c.v.) infusion of insulin (12 mU/day in 2.4 ml saline) or saline (2.4 ml/day) for 5 days (n = 6) in a crossover design. The dose of insulin (i.c.v.) was calculated to approximate the increase in CSF insulin concentration found after peripheral insulin treatment. To monitor LH secretory patterns, blood samples were collected by jugular venipuncture at 10-min intervals for 4 h on the day before and 5 days after the start of i.c.v. insulin infusion. To monitor the increase in CSF insulin concentrations, a single CSF sample was collected one and four days after the start of the central infusion. The i.c.v. insulin infusion increased CSF insulin concentrations above those in saline-treated animals (P < 0.05) and maintained them at or above the peak levels achieved after peripheral insulin treatment. Central insulin infusion did not affect peripheral (plasma) insulin or glucose concentrations. LH pulse frequency in insulin-treated animals was greater than that in saline-treated animals (3.5 +/- 0.2 vs. 2.3 +/- 0.3 pulses/4 h, P < 0.01), but it was less than that during peripheral insulin treatment (4.8 +/- 0.2 pulses/4 h, P < 0.01). Our findings suggest that physiologic levels of central insulin supplementation are able to increase pulsatile LH secretion in diabetic sheep with low peripheral insulin. These results are consistent with the notion that central insulin plays a role in regulating pulsatile GnRH secretion.     

Murine islet cryopreservation and corticosteroids: functional studies

Knowledge of protective effects of corticosteroids on traumatized cells prompted us to test the potential benefit of islet cryopreservation in the presence of hydrocortisone. Neonatal murine islets were isolated by collagenase, followed by 2- to 3-day tissue culture. Precryopreservation glucose-stimulated (50-500 mg/dl) insulin release was 25-388% above basal (mean = 113%) in 18/20 fresh islet preparations. Subsequent freezing was done in RPMI 1640 medium plus 10% (v/v) heat-inactivated fetal calf serum and 10% (v/v) Me2SO with or without 1 mg/ml hydrocortisone at 0.25 degrees C per minute in a programmed freezing system, to -80 degrees C, and stored for greater than 60 days at -196 degrees C. Thawing, by transfer to room air, was followed by dilution, 4x (v/v), in 4 degrees C RPMI plus 10% protein, after which glucose-stimulated insulin release was reassessed, showing 56-280% response over basal in 3/8 steroid-treated preparation and 20-220% response in 3/10 control preparations. Basal insulin release was 0.72 ng/microgram protein/hr in fresh islets (N = 20) and 0.22 ng/microgram protein/hr after freeze-thawing. We conclude that functional islet survival by this method is approximately 30% and that hydrocortisone did not improve viability.     

A novel drug to reduce tumor perfusion: antitumor effect alone and with hyperthermia

We have investigated the feasibility of enhancing damage induced by hyperthermia in SCK murine tumors by reducing tumor blood perfusion using a new agent, KB-R8498. Within several minutes of an i.v. injection, the tumor perfusion was reduced to less than 20% of the control value, and it recovered to 40-70% of the control value by 1 h after injection. The perfusion in normal tissues decreased or increased soon after drug administration depending on the tissue type. However, by 1 h after drug treatment, perfusion in five of the seven tissues examined had returned to the control level. The tumor pH was also reduced after i.v. drug administration. Control tumors grew to four times the initial volume in 6 days. Tumors that were heated at 42.5 degrees C for 60 min were delayed in growth by 4 days compared to control tumors. There was a growth delay of 14 days when an i.v. injection of KB-R8498 was given and the tumors were heated at 42.5 degrees C either immediately or 1 h later. In drug-alone studies, the tumor growth was delayed by 4 days when the drug was infused continuously at a rate of 30-50 mg/kg day(-1) for 7 days or about 2 days when mice were treated with five daily injections of 30 mg/kg KB-R8498.     

Insulin bolus given by sprinkler needle: effect on absorption and glycaemic response to a meal.

Two studies were conducted investigating the effect of injecting short acting insulin subcutaneously by means of a sprinkler needle; this needle has 14 small holes in the wall but is sealed at the tip. In the first study absorption of 8 U iodine-125 labelled Actrapid HM injected subcutaneously at two separate sites in the abdominal wall was measured in 10 patients. One injection was given with the sprinkler needle and the other with a conventional needle. The initial dose absorbed during the first 30 minutes was significantly higher with the sprinkler needle. In the second study 10 U Actrapid HM was given to 11 other patients (all negative for C peptide and with low insulin binding antibody titres) on two separate days immediately before a standardised breakfast either by the sprinkler needle or by a conventional needle (random order). Plasma free insulin increased more rapidly and to higher concentrations with the sprinkler needle and the glycaemic response was considerably diminished. The sprinkler needle improves both the insulin absorption rate and the glycaemic response to a meal and may reduce the 30 minutes or so before meals that diabetics must inject to minimise postprandial hyperglycaemia.     

Effect of space flight and head-down bedrest on neuroendocrine response to metabolic stress in physically trained subjects

The aim of this study was to evaluate the association of plasma epinephrine (EPI) and norepinephrine (NE) responses to insulin induced hypoglycemia (ITT) 3 weeks before the space flight (SF), on the 5th day of SF, on the 2nd and 16th days after the landing in the first Slovak astronaut, and before and on the 5th day of prolonged subsequent head-down (-6 degrees) bed rest (BR) in 15 military aircraft pilots. Blood samples during the test were collected via cannula inserted into cubital vein, centrifuged in the special appliance Plasma-03, frozen in Kryogem-03, and at the end of the 8-day space flight transferred to Earth in special container for hormonal analysis. Insulin hypoglycemia was induced by i.v. administration of 0.1 IU/kg BW insulin (Actrapid HM) in bolus. Insulin administration led to a comparable hypoglycemia in pre-flight, in-flight conditions and before and after bed rest. ITT led to a pronounced increase in EPI levels and moderate increase in NE in pre-flight studies. However, an evidently reduced EPI response was found after insulin administration during SF and during BR. Thus, during the real microgravity in SF and simulated microgravity in BR, insulin-induced hypoglycemia activates the adrenomedullary system to less extent than at conditions of the Earth gravitation. Post-flight changes in EPI and NE levels did not significantly differ from those of pre-flight since SF was relatively short (8 days) and the readaptation to Earth gravitation was fast. It seems, that an increased blood flow in brain might be responsible for the reduced EPI response to insulin. Responses to ITT in physically fit subjects indicate the stimulus specificity of deconditioning effect of 5 days bed rest on stress response. Thus, the data indicate that catecholamine responses to ITT are reduced after exposure to real as well as simulated microgravity.     

Lifespan of subcutaneous glucose sensors and their performances during dynamic glycaemia changes in rats

Performances of a glucose sensor have been investigated during dynamic variations of plasma glucose levels. Subcutaneous glucose concentrations measured by the sensors were calculated by a one-point calibration, performed in basal conditions. A first group of sensors were chronically implanted in the subcutaneous tissue of normal rats. The animals were submitted to glucagon and insulin injection, in order to induce rapid modifications of their glycaemia. This test was repeated at different days after implantation in order to investigate the lifespan and the performance of the sensors. All the sensors were working 1 or 2 days after implantation, and 70% adequately responded to glycaemia variations at day 3 or 4. The quality of the sensors' performance remained constant as a function of the time. With a second group of sensors, we demonstrated that an efficient sterilization procedure did not alter the sensors' characteristics. At the day of implantation, the sterilized sensors' performance, during dynamic variations of plasma glucose levels, was closely similar to that of the non-sterilized sensors. The animals bearing the sterilized devices were rendered diabetic by steptozotocin (STZ) injection. Once the rats had developed a severe hyperglycaemia (1–3 days after STZ), they were injected with intravenous insulin. The subcutaneously implanted glucose sensors correctly followed the decline in plasma glucose levels. We therefore conclude that our sensor could represent a useful tool for short-term continuous blood monitoring.     

Changes in pituitary responses to synthetic ovine corticotrophin releasing factor in fetal sheep

The rise in cortisol in fetal sheep during late pregnancy has been related to increased responsiveness of the adrenal to ACTH. Most reports have suggested that plasma ACTH concentrations rise coincident with or after the prepartum increase in cortisol. To reexamine the relationship of cortisol with basal immunoreactive ACTH (IR-ACTH) throughout the last 40 days of pregnancy and to determine changes in fetal pituitary responsiveness during this time, we measured basal and synthetic ovine corticotrophin-releasing factor (oCRF) (10 ng-10 micrograms) induced rises in ACTH and cortisol in fetal sheep at days 110-115, 125-130, and 135-140 of pregnancy. The fetuses were catheterized on day 105-120 and entered spontaneous labour at greater than 140 days. Basal IR-ACTH (picograms per millilitre +/- SEM) rose from 16.7 +/- 2.9 pg/mL at day 110-115 to 34.8 +/- 8.7 pg/mL at day 141-145. There was a significant effect of time on basal ACTH concentrations with a mean increase of approximately 5 pg ACTH per millilitre of plasma per 5-day sampling interval. Plasma cortisol changed gradually between day 110 and 125 of gestation and then more rapidly to term. At day 110-115 of gestation there was no significant change in plasma ACTH after 10 or 100 ng oCRF, but there was a significant increase in ACTH after 1 microgram of oCRF. Plasma cortisol did not change after any CRF injection. The change in IR-ACTH after oCRF at day 125-130 of gestation was significantly greater than that at day 110-115. Plasma cortisol concentrations were elevated following 1- and 10-micrograms injections of oCRF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variability of Insulin Action: Does It Matter?

Background: Repeat SC injection of identical insulin doses does not induce an identical metabolic effect in patients with diabetes. This concept hampers practical insulin therapy tremendously.Objective: The aim of this article is to briefly and critically review the available literature on the variability of insulin action.Methods: There are only limited numbers of clinical-experimental and clinical studies focusing on the variability of insulin action; therefore, no formal literature search was performed. However, the available studies, along with their references, provided the basis for our findings.Results: Insulin absorption from the SC depot is mainly determined by local blood flow, and many factors affect blood flow. The metabolic effect of absorbed insulin depends on a patient's insulin sensitivity, which is highly variable both intraindividually and interindividually. SC injection of prandial insulin shows an intraindividual coefficient of variation (CV) of insulin action of 15% to 25% and an interindividual CV of 20% to 45% under experimental conditions. The intraindividual variability of insulin action for intermediate-acting/basal insulin is not much higher at 25% to 35% than that of prandial insulin. The variability of the currently available soluble long-acting insulin analogues is reduced compared with neutral protamine Hagedorn insulin in most but not all studies. Although other routes of insulin application (eg, inhalation) might provide an opportunity to reduce the variability of insulin action, the intraindividual variability of metabolic effect observed after inhalation of insulin (15%–30%) was comparable to that seen after SC administration of prandial insulin (15%–25%).Conclusions: The variability of insulin action is clinically highly relevant; however, an intensive scientific investigation of this topic is lacking. Nonetheless, this research might well enable the development of insulin formulations or insulin application techniques that would help reduce this disturbing aspect of insulin therapy.     

Comparison of the subcutaneous absorption of insulin glargine (Lantus) and NPH insulin in patients with Type 2 diabetes.

The aim of this study was to compare the subcutaneous absorption characteristics of insulin glargine with NPH insulin in patients with Type 2 diabetes. In this single-dose, double-blind, randomized, two-way crossover study, 14 patients with Type 2 diabetes (aged 40-70 years) previously untreated with insulin were randomized to receive in a fasting state either a single subcutaneous injection of 0.3 U/kg 125I-insulin glargine or 0.3 U/kg 125I-NPH insulin. The disappearance of radioactivity was monitored for forty-eight hours. The median time for 25%, 50% and 75% of the radioactivity to disappear from the injection site was significantly longer for insulin glargine compared with NPH insulin (T75% 15.0 and 6.5 h, p=0.009; T50% 26.3 and 13.4 h, p=0.009; T25% 42.4 and 26.6 h, p=0.019, respectively). The mean residual radioactivity remaining at 24, 36 and 48 h after injection remained significantly higher than NPH insulin (54.4 and 27.9%, p=0.0001; 35.0 and 17.0%, p=0.003; 19.2 and 9.2%, p=0.01, respectively). Mean plasma glucose levels reached a minimum after 14.6 and 9 h in response to insulin glargine and NPH insulin, respectively. The subcutaneous absorption of insulin glargine in fasting Type 2 diabetes patients was significantly (2-3 times) slower compared with NPH insulin in patients with Type 2 diabetes. The slower absorption of insulin glargine correlated with the fall in plasma glucose levels over a 24 h period compared with the faster insulin absorption and more rapid decrease in plasma glucose levels observed in response to NPH insulin. Both insulin glargine and NPH insulin were well tolerated.     

Complex of recombinant heat shock protein with lipopolysaccharide induces rapid protection of mice against Salmonella typhimurium and avirulent for humans avian influenza virus H5N2

Protective effect of immunization with heat shock protein (HSP) against bacterial and viral infections in mice was studied. Recombinant HSP 70 kDa of Mycobacterium tuberculosis contaminated with lypopolysaccharide (0.185 mcg/ml) was used for experiments. One intraperitoneal injection of 100 or 400 mcg of HSP induced rapid protection against intraperitoneal challen e with 125 LD50 of Salmonella typhimurium (on 3rd-6th day) and against intranasal challenge with 10 LD50 of avirulent for humans avian influenza virus H5N2 (A/ mallard/Pennsylvania/10218/84) (on 5th-8th day). Three daily injections with 10 mcg of HSP induced rapid, significant and long-term protection against S. typhimurium. Immunization with HSP protected 100% of mice during 3 days after the challenge, 50% of immunized animals survived during 21 days (duration of the study). All nonimmunized mice died on 6th day.     

The effect of fasting on the activation in vivo of the insulin receptor kinase.

Fasting causes insulin resistance in liver and fat, and increases insulin sensitivity in muscle. We studied the response in vitro and in vivo to insulin of the insulin receptor tyrosine kinase in muscle and liver from 72 h fasted and control rats. Insulin was injected intraperitoneally together with glucose, and blood and tissue samples were obtained 0, 5, 15 and 30 min later. Basal serum glucose and insulin levels were significantly higher in control than in fasting rats. Serum glucose rose to approximately 300 mg/dl at 5 min and then progressively declined without hypoglycaemia. Receptors were prepared from whole tissue by wheat germ lectin affinity chromatography. 125I-insulin binding to purified receptors was increased by fasting in both muscle (18%) and liver (50%). In untreated fasting and control animals, muscle and liver insulin receptor tyrosine kinase activity was stimulated to similar levels by insulin added in vitro. With only insulin treatment in vivo, muscle receptor tyrosine kinase behaved similarly in fasting and control animals with maximal activation at 15 min post injection. In liver, insulin in vivo stimulated receptor tyrosine kinase activity maximally at 5 min post injection in both fasting and control, but in fasting animals the treatment in vivo caused a significantly larger and more prolonged activation of the enzymic activity, possibly due to a decrease in the rate of dephosphorylation and deactivation of the beta subunits.     

Influence of chronic treatment with iopanoic acid on thyroxine metabolism in the newborn rat

Parameters of the peripheral metabolism of thyroxine (T4) were studied in the early postnatal period. Iopanoic acid (IOP) was administered to newborn rats that were either euthyroid or rendered hypothyroid in utero by propylthiouracil (PTU) or methimazole (MMI) administration to the mothers during gestation and injected with thyroxine on postnatal days 6 and 7. In euthyroid newborn rats given IOP from postnatal day 6, the plasma T4 level increased (+50%) while the plasma 3,3',5'-triiodothyronine (T3) level slightly decreased (-18%). Peripheral deiodination of T4 was also reduced (about -50%) as estimated by thyroid 125I uptake after injection of 125I (3'-5')L-T4. In the newborn rats rendered hypothyroid in utero and given T4 on postnatal days 6 and 7, IOP treatment started on day 4 decreased the constant rate of elimination (-50%), the distribution volume (-43%) and the metabolic clearance (-74%) of plasma T4. The results were the same in PTU- and MMI-treated newborn rats. The differences between newborn and adult animals under IOP treatment are discussed.     

Cell-surface insulin receptor cycling and its implication in the glycogenic response in cultured foetal hepatocytes.

The insulin-receptor cycle was investigated in cultured foetal rat hepatocytes by determining the variations in insulin-binding sites at the cell surface after short exposure to the hormone. Binding of 125I-insulin was measured at 4 degrees C after dissociation of prebound native insulin. Two protocols were used: exchange binding assay and binding after acid treatment; both gave the same results. Cell-surface 125I-insulin-receptor binding decreased sharply (by 40%) during the first 5 min of 10 nM-insulin exposure (t1/2 = 2 min) and remained practically constant thereafter; subsequent removal of the hormone restored the initial binding within 10 min. This fall-rise sequence corresponded to variations in the number of insulin receptors at the cell surface, with no detectable change in receptor affinity. The reversible translocation of insulin receptors from the cell surface to a compartment not accessible to insulin at 4 degrees C was hormone-concentration- and temperature-dependent. SDS/polyacrylamide-gel electrophoresis after cross-linking of bound 125I-insulin to cell-surface proteins with disuccinimidyl suberate showed that these variations were not associated with changes in Mr of binding components, in particular for the major labelled band of Mr 130,000. The insulin-receptor cycle could be repeated after intermittent exposure to insulin. Continuous or intermittent exposure to the hormone gave a similar glycogenic response, contrary to the partial effect of a unique short (5-20 min) exposure. A relationship could be established between the repetitive character of the rapid insulin-receptor cycle and the maximal expression of the biological effect in cultured foetal hepatocytes.     

Administration of semisynthetic human insulin by a spray injector

The use of Jet injection in insulin administration pointed out the question whether this route could affect insulin absorption and plasma insulin profiles. To compare plasma insulin profiles following an administration of an identical insulin dose by jet injection or by conventional subcutaneous route (syringe with needle) 8 healthy subjects (age 24-28 yrs., non obese) were given at 09.00 h of two different days 200 mU/kg/BW of human semisynthetic regular insulin (Novo Actarapid) alternatively subcutaneously by a syringe with needle or transcutaneously by jet injection (DG 77 - Sicim - Gorizia). Before insulin administration and then 15, 30, 60, 90, 120 and 180 minutes after, blood samples were drawn for plasma insulin and C-peptide determination. Higher plasma insulin levels after administration by jet were found at 15' and 30' minutes (62,58 +/- 6,31 v.s. 36,94 +/- 3,31 microunits/ml at 15' and 76,51 +/- 9,60 v.s. 51,65 +/- 9,95 at 30', p less than 0,01 and p less than 0,005, paired Student t test). No difference could be observed for the other times. C-peptide was found to fall to undetectable values, confirming the nearly total suppression of endogenous insulin production. It is concluded that total regular insulin absorption does not differ after transcutaneous jet injection or administration by syringe with needle, but in the first case it is faster. This last finding should be considered in planning insulin treatment schedules.     

Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture.

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Pentagastrin-induced gastric acid secretion in the diabetic rats: role of insulin

Streptozotocin-induced diabetic rats have excessively pentagastrin-simulated acid output in which insulin seems to attenuate rather than further stimulate acid output. The aim of this study was to determine the insulin impact on pentagastrin-stimulated acid output of diabetic and non-diabetic rats to resolve whether an attenuated effect does exist. Diabetic rats were induced by the streptozotocin i.v. injection four days before acid study. Some streptozotocin-treated rats additionally received daily insulin (2.4 IU/kg) injection. Using an autotitrator, acid output was measured every five minutes by the titration of gastric perfusate. Basal output was collected for 45 min before the 90-min pentagastrin infusion (0.89 microg/kg/min). Plasma gastric inhibitory polypeptide (GIP) levels were measured. Both doses (0.067 and 0.133 IU/kg/min) of insulin infusion resulted in stimulated acid output in normal rats. The subsequent insulin infusion (0.133 IU/kg/min) for non-diabetic rats undergoing pentagastrin-treatment suppressed their stimulated acid output almost down to the basal level. Pentagastrin-stimulation led to the excessively increased acid output of diabetic rats throughout the whole infusion period (P < 0.01). Correction of hyperglycemia with insulin for diabetic rats normalized the stimulated acid output. Measured basal and stimulated plasma GIP levels of those diabetic rats during acid stimulation remained higher, regardless of insulin treatment (P < 0.05). Our results suggest that insulin has the ability to attenuate pentagastrin-stimulated acid output in rats, whereas GIP is not involved in this attenuation. This effect appears to be responsible for the excessive acid output of diabetic rats undergoing pentagastrin stimulation.     

Antihyperalgesic effect of simultaneously released hydromorphone and bupivacaine from polymer fibers in the rat chronic constriction injury model

We aimed to evaluate the antihyperalgesic efficacy of a combination of hydromorphone (HM) and bupivacaine (BP) delivered via controlled release from a biodegradable cylindrical rod. In vivo studies were performed using a rat model of thermal hyperalgesia induced by chronic constriction injury (CCI) of the sciatic nerve with loose ligatures. Poly(lactic-co-glycolic acid) (PLGA) rods (10 mm length, 1 mm diameter) loaded with HM (5 mg per rod), BP (5 mg per rod) or no drug (placebo) were implanted subcutaneously, in single or dual pairs, adjacent to the constriction injury, immediately after nerve ligation. We evaluated the efficacy of two dose levels for each drug, alone or in combination, in attenuating thermal hyperesthesia over a period of 12 days according to a prevention protocol. Plasma levels of drugs released from the rods and also released in an in vitro simulation were evaluated. In vitro studies demonstrated that drug release is maintained for at least 10 days. HM (5 mg) alone and BP (5 mg) alone did not attenuate hyperalgesia. Their combination provided a significant increase in the paw withdrawal latency as compared to single agents or placebo. When the dose in each group was doubled, implanting four rods, significant attenuation of hyperalgesia was observed. Analyses of rods retrieved after termination of experiments (after 12 days) revealed 30% residual HM and 70% residual BP content. Prolonged delivery of HM and BP alone or in combination via locally applied PLGA rods may offer a feasible alternative to provide long-lasting analgesia.