Brian L. Furman

Reactive oxygen species and endothelial function in diabetes.

An increasing body of evidence suggests that oxidant stress is involved in the pathogenesis of many cardiovascular diseases, including hypercholesterolemia, atherosclerosis, hypertension, heart failure and diabetes. Recent studies have also provided important new insights into potential mechanisms underlying the pathogenesis of vascular disease induced by diabetes. Glycosylation of proteins and lipids, which can interfere with their normal function, activation of protein kinase C with subsequent alteration in growth factor expression, promotion of inflammation through the induction of cytokine secretion and hyperglycemia-induced oxidative stress are some of these mechanisms. It is widely accepted that hyperglycemia-induced reactive oxygen species contribute to cell and tissue dysfunction in diabetes. A variety of enzymatic and non-enzymatic sources of reactive oxygen species exist in the blood vessels. These include NADPH oxidase, mitochondrial electron transport chain, xanthine oxidase and nitric oxide synthase. The present article reviews the effects of reactive oxygen species on endothelial function in diabetes and addresses possible therapeutic interventions.

Cyclic nucleotide phosphodiesterases in pancreatic islets

Cyclic nucleotide phosphodiesterases (PDEs) comprise a family of enzymes (PDE1-PDE11) which hydrolyse cyclic AMP and cyclic GMP to their biologically inactive 5′ derivatives. Cyclic AMP is an important physiological amplifier of glucose-induced insulin secretion. As PDEs are the only known mechanism for inactivating cyclic nucleotides, it is important to characterise the PDEs present in the pancreatic islet beta cells. Several studies have shown pancreatic islets or beta cells to contain PDE1C, PDE3B and PDE4, with some evidence for PDE10A. Most evidence suggests that PDE3B is the most important in relation to the regulation of insulin release, although PDE1C could have a role. PDE3-selective inhibitors augment glucose-induced insulin secretion. In contrast, activation of beta-cell PDE3B could mediate the inhibitory effect of IGF-1 and leptin on insulin secretion. In vivo, although PDE3 inhibitors augment glucose-induced insulin secretion, concomitant inhibition of PDE3B in liver and adipose tissue induce insulin resistance and PDE3 inhibitors do not induce hypoglycaemia. The development of PDE3 inhibitors as anti-diabetic agents would require differentiation between PDE3B in the beta cell and that in hepatocytes and adipocytes. Through their effects in regulating beta-cell cyclic nucleotide concentrations, PDEs could modulate beta-cell growth, differentiation and survival; some work has shown that selective inhibition of PDE4 prevents diabetes in NOD mice and that selective PDE3 inhibition blocks cytokine-induced nitric oxide production in islet cells. Further work is required to understand the mechanism of regulation and role of the various PDEs in islet-cell function and to validate them as targets for drugs to treat and prevent diabetes.

Ryanodine receptors of pancreatic beta-cells mediate a distinct context-dependent signal for insulin secretion

The ryanodine (RY) receptors in β‐cells amplify signals by Ca2+‐induced Ca2+ release (CICR). The role of CICR in insulin secretion remains unclear in spite of the fact that caffeine is known to stimulate secretion. This effect of caffeine is attributed solely to the inhibition of cAMP‐phosphodiesterases (cAMP‐PDEs). We demonstrate that stimulation of insulin secretion by caffeine is due to a sensitization of the RY receptors. The dose‐response relationship of caffeine‐induced inhibition of cAMP‐PDEs was not correlated with the stimulation of insulin secretion. Sensitization of the RY receptors stimulated insulin secretion in a context‐dependent manner, that is, only in the presence of a high concentration of glucose. This effect of caffeine depended on an increase in [Ca2+]i. Confocal images of β‐cells demonstrated an increase in [Ca2+]i induced by caffeine but not by forskolin. 9‐Methyl‐7‐bromoeudistomin D (MBED), which sensitizes RY receptors, did not inhibit cAMP‐PDEs, but it stimulated secretion in a glucose‐dependent manner. The stimulation of secretion by caffeine and MBED involved both the first and the second phases of secretion. We conclude that the RY receptors of β‐cells mediate a distinct glucose‐dependent signal for insulin secretion and may be a target for developing drugs that will stimulate insulin secretion only in a glucose‐dependent manner.

Streptozotocin‐Induced Diabetic Models in Mice and Rats

Streptozotocin (STZ) is an antibiotic that produces pancreatic islet β‐cell destruction and is widely used experimentally to produce a model of type 1 diabetes mellitus (T1DM). Detailed in this unit are protocols for producing STZ‐induced insulin deficiency and hyperglycemia in mice and rats. Also described are protocols for creating animal models for type 2 diabetes using STZ. These animals are employed for assessing the pathological consequences of diabetes and for screening potential therapies for the treatment of this condition.

Delayed protection against ischaemia-induced ventricular arrhythmias and infarct size limitation by the prior administration of Escherichia coli endotoxin.

1 Bacterial endotoxin (lipopolysaccharide derived from Escherichia coli) was injected intraperitoneally in conscious rats in doses ranging from 0.5 to 2.5 mg kg−1. At various times afterwards the animals were anaesthetized and subjected to a 30 min period of left coronary artery occlusion. 2 Under these conditions the severity of ventricular arrhythmias was markedly suppressed, in comparison with saline‐injected controls, but this was particularly marked with the higher doses (1.5 and 2.5 mg kg−1); the number of ventricular premature beats was reduced from 1687 ± 227 over the 0.5 h coronary artery occlusion period to 190 ± 46 in those rats administered 2.5 mg kg−1 endotoxin 8 h previously (P < 0.05). The duration of ventricular tachycardia was also significantly reduced (138 ± 26 s to 8.9 ± 4.2 s; P < 0.01) and there was a reduction in the incidence of ventricular fibrillation (from 56% to 10%). 3 The time course of this protection was studied following the administration of a single dose of 2.5 mg kg−1 of endotoxin by anaesthetizing rats 4, 8 or 24 h later. Protection was apparent at each time but was particularly marked at 8 h. 4 No rat given the highest dose of endotoxin (32 in all) died as a result of ventricular fibrillation, or from any other cause, during an occlusion, in contrast to a 26% mortality in the controls (P < 0.01). 5 Infarct size, measured following a 30 min period of coronary artery occlusion followed by a 3 h reperfusion period, was reduced both 8 and 24 h after the administration of 2.5 mg kg−1 endotoxin (reductions of 24.3 and 23.1% respectively; P < 0.05). Endotoxin had no significant effect on the area at risk. 6 The beneficial effects of endotoxin on infarct size and on ventricular arrhythmias were markedly attenuated by the prior administration of dexamethasone, 3 mg kg−1 given 1 h prior to endotoxin administration. Dexamethasone itself reduced infarct size (P < 0.05) but had no direct effect on arrhythmia severity following coronary artery occlusion. 7 The mechanisms of this ‘cross‐tolerance’ induced by bacterial endotoxin against ischaemia‐reperfusion injury remain to be elucidated but the most likely mechanisms appear to be the induction of protective enzymes or proteins (e.g. nitric oxide synthase, cyclo‐oxygenase (COX) 2) probably mediated by cytokine release.

Endotoxin-induced impairment of vasopressor and vasodepressor responses in the pithed rat.

1 Effects of E. coli endotoxin on vascular responsiveness to a variety of agents were compared with those of the calcium channel blocking drug nicardipine in pithed rats. 2 Infusion of endotoxin (250 μg kg−1 h−1) produced a fall in mean arterial blood pressure (8 mmHg). A similar fall (11 mmHg) was seen in rats receiving nicardipine (1.0 mg kg−1). 3 Endotoxin impaired responsiveness to vasopressin, phenylephrine and cirazoline, producing a shift to the right in the dose‐response curves without any change in the maximum response. Responsiveness to 5‐hydroxytryptamine (5‐HT) and to the α2‐adrenoceptor agonists clonidine and BHT 933, was also impaired with a marked reduction in their maximum responses. The dose‐response curve to the pressor effects of endothelin was not significantly modified. 4 Nicardipine produced a similar pattern of impairment of responsiveness to these agents to that produced by endotoxin. However, nicardipine also shifted the pressor dose‐response curve to endothelin to the right with no significant alteration in its maximum response. 5 The pressor responses to endothelin and to 5‐HT were, respectively, preceded and followed by dose‐dependent depressor responses, which were markedly reduced by endotoxin and nicardipine. 6 The concomitant infusion of arginine vasopressin (0.64 iu kg−1 h−1) prevented endotoxin‐induced hypotension and also prevented the impairment in responsiveness to cirazoline and to BHT 933. 7 The similarity of the pattern of impaired pressor responsiveness (except in relation to endothelin) and depressor responsiveness produced by endotoxin and nicardipine may be consistent with a common mechanism of action.

Effects of type‐selective phosphodiesterase inhibitors on glucose‐induced insulin secretion and islet phosphodiesterase activity

1 We examined various type‐selective phosphodiesterase (PDE) inhibitors on glucose‐induced insulin secretion from rat isolated islets, on islet PDE activity and on islet cyclic AMP accumulation in order to assess the relationship between type‐selective PDE inhibition and modification of insulin release. 2 The nonselective PDE inhibitor, 3‐isobutyl‐l‐methylxanthine (IBMX, 10−5‐10−3 M), as well as the type III selective PDE inhibitors SK&F 94836 (10−5‐10−3 M), Org 9935 (10−7‐10−4 M), SK&F 94120 (10−5‐10−4 M) and ICI 118233 (10−6‐10−4 M) each caused concentration‐dependent augmentation (up to 40% increase) of insulin release in the presence of a stimulatory glucose concentration (10 mM), but not in the presence of 3 mM glucose. 3 Neither the type IV PDE inhibitor rolipram (10−4 M) nor the type I and type V PDE inhibitor, zaprinast (10−4‐10−3 M) modified glucose‐induced insulin release when incubated with islets, although a higher concentration of rolipram (10−3 M) inhibited secretion by 55%. However, when islets were preincubated with these drugs followed by incubation in their continued presence, zaprinast (10−6‐10−4 M) produced a concentration‐dependent inhibition (up to 45% at 10−4 M). Under these conditions, rolipram inhibited insulin secretion at a lower concentration (10−4 M) than when simply incubated with islets. 4 A combination of SK&F 94836 (10−5 M) and forskolin (5 × 10−8 M) significantly augmented glucose‐induced insulin secretion (30% increase), although neither drug alone, in these concentrations, produced any significant effect. 5 Islet cyclic AMP levels, which were not modified by forskolin (10−6 M), SK&F 94836 (10−4 M) or Org 9935 (10−5 M) were significantly elevated (approximately 3.7 fold increase) by forskolin in combination with either SK&F 94836 or Org 9935. 6 Homogenates of rat islets showed a low Km (1.7 μm) and high Km (13 μm) cyclic AMP PDE in the supernatant fractions (from 48, 000 g centrifugation), whereas the particulate fraction showed only a low Km (1.4 μm) cyclic AMP PDE activity. 7 The PDE activity of both supernatant and pellet fractions were consistently inhibited by SK&F 94836 or Org 9935, the concentrations required to reduce particulate PDE activity by 50% being 5.5 and 0.05 μm respectively. 8 Rolipram (10−5‐10−4 M) did not consistently inhibit PDE activity in homogenates of rat islets and zaprinast (10−4 M) consistently inhibited activity by 30% in the supernatant fraction, but not consistently in the pellet. 9 These data are consistent with the presence of a type III PDE in rat islets of Langerhans.

Effects of inhibitors of 5-hydroxytryptamine uptake on plasma glucose and their interaction with 5-hydroxytryptophan in producing hypoglycaemia in mice

The effects of various inhibitors of 5-HT uptake on plasma glucose have been studied in normal and monoamine oxidase inhibitor pretreated mice. Additionally their interaction with 5-hydroxytryptophan (5-HTP) in producing hypoglycaemia was studied. Clomipramine, fenfluramine, fluoxetine, ORG 6582 (dl-8-chloro-11-anti-amino-benzo-(b)-bicyclo[3.3.1]nona-3, 6 alpha(10 alpha)-diene hydrochloride), ORG 6997 (dl-4-exo-amino-8-chloro-benzo-(b)-bicyclo[3.3.1]nona-2-6 alpha(10 alpha)-diene hydrochloride), MK 212 and mazindol did not modify the plasma glucose in normal mice but produced hypoglycaemia in mice pretreated with either nialamide or pargyline. Dexamphetamine did not influence plasma glucose in either normal or monoamine oxidase inhibitor pretreated mice. Each of the above drugs except ORG 6997 but including dexamphetamine augmented the hypoglycaemic effect of 5-HTP in normal mice. These responses did not appear to be mediated by insulin since none of the drugs increased the plasma immunoreactive insulin concentration or augmented the hyperinsulinaemic effect of 5-HTP. Moreover, fenfluramine, fluoxetine and ORG 6582 did not augment the hypoglycaemic action of injected insulin although such an augmentation was produced by mazindol.

Mechanisms underlying the metabolic actions of galegine that contribute to weight loss in mice

Background and purpose: Galegine and guanidine, originally isolated from Galega officinalis, led to the development of the biguanides. The weight‐reducing effects of galegine have not previously been studied and the present investigation was undertaken to determine its mechanism(s) of action.

Targeting beta-cell cyclic 3 ` 5 ` adenosine monophosphate for the development of novel drugs for treating type 2 diabetes mellitus. A review

Cyclic 3′5′AMP is an important physiological amplifier of glucose‐induced insulin secretion by the pancreatic islet β‐cell, where it is formed by the activity of adenylyl cyclase, especially in response to the incretin hormones GLP‐1 (glucagon‐like peptide‐1) and GIP (glucose‐dependent insulinotropic peptide). These hormones are secreted from the small intestine during and following a meal, and are important in producing a full insulin secretory response to nutrient stimuli. Cyclic AMP influences many steps involved in glucose‐induced insulin secretion and may be important in regulating pancreatic islet β‐cell differentiation, growth and survival. Cyclic AMP (cAMP) itself is rapidly degraded in the pancreatic islet β‐cell by cyclic nucleotide phosphodiesterase (PDE) enzymes. This review discusses the possibility of targeting cAMP mechanisms in the treatment of type 2 diabetes mellitus, in which insulin release in response to glucose is impaired. This could be achieved by the use of GLP‐1 or GIP to elevate cAMP in the pancreatic islet β‐cell. However, these peptides are normally rapidly degraded by dipeptidyl peptidase IV (DPP IV). Thus longer‐acting analogues of GLP‐1 and GIP, resistant to enzymic degradation, and orally active inhibitors of DPP IV have also been developed, and these agents were found to improve metabolic control in experimentally diabetic animals and in patients with type 2 diabetes. The use of selective inhibitors of type 3 phosphodiesterase (PDE3B), which is probably the important pancreatic islet β‐cell PDE isoform, would require their targeting to the islet β‐cell, because inhibition of PDE3B in adipocytes and hepatocytes would induce insulin resistance.