Preprandial versus postprandial blood glucose monitoring in type 1 diabetic pregnancy: A randomized controlled clinical trial

OBJECTIVE: This study was undertaken to compare preprandial and postprandial capillary glucose monitoring in pregnant women with type 1 diabetes.

Effects of sub-chronic exposure to naturally occurring N-terminally truncated metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), GIP(3-42) and GLP-1 (9-36)amide, on insulin secretion and glucose homeostasis in ob/ob mice

Glucose-dependent insulinotrophic polypepticle (GIP) and glucagon-like peptide-1 (GLP-1) are important enteroendocrine hormones that are rapidly degraded by an ubiquitous enzyme dipeptidyl peptidase IV to yield truncated metabolites GIP(3-42) and GLP-1 (9-36)amide. In this study, we investigated the effects of sub-chronic exposure to these major circulating forms of GIP and GLP-1 on blood glucose control and endocrine pancreatic function in obese diabetic (ob/ob) mice. A once daily injection of either peptide for 14 days had no effect on body weight, food intake or pancreatic insulin content or islet morphology. GLP-1(9-36)amide also had no effect on plasma glucose homeostasis or insulin secretion. Mice receiving GIP(3-42) exhibited small but significant improvements in non-fasting plasma glucose, glucose tolerance and glycaemic response to feeding. Accordingly, plasma insulin responses were unchanged suggesting that the observed enhancement of insulin sensitivity was responsible for the improvement in glycaemic control. These data indicate that sub-chronic exposure to GIP and GLP-1 metabolites does not result in physiological impairment of insulin secretion or blood glucose control. GIP(3-42) might exert an overall beneficial effect by improving insulin sensitivity through extrapancreatic action.

Inhibition of Advanced Glycation and Absence of Galectin-3 Prevent Blood-Retinal Barrier Dysfunction during Short-Term Diabetes

Breakdown of the inner blood-retinal barrier (iBRB) occurs early in diabetes and is central to the development of sight-threatening diabetic macular edema (DME) as retinopathy progresses. In the current study, we examined how advanced glycation end products (AGEs) forming early in diabetes could modulate vasopermeability factor expression in the diabetic retina and alter inter-endothelial cell tight junction (TJ) integrity leading to iBRB dysfunction. We also investigated the potential for an AGE inhibitor to prevent this acute pathology and examined a role of the AGE-binding protein galectin-3 (Gal-3) in AGE-mediated cell retinal pathophysiology. Diabetes was induced in C57/BL6 wild-type (WT) mice and in Gal-3(-/-) transgenic mice. Blood glucose was monitored and AGE levels were quantified by ELISA and immunohistochemistry. The diabetic groups were subdivided, and one group was treated with the AGE-inhibitor pyridoxamine (PM) while separate groups of WT and Gal-3(-/-) mice were maintained as nondiabetic controls. iBRB integrity was assessed by Evans blue assay alongside visualisation of TJ protein complexes via occludin-1 immunolocalization in retinal flat mounts. Retinal expression levels of the vasopermeability factor VEGF were quantified using real-time RT-PCR and ELISA. WT diabetic mice showed significant AGE -immunoreactivity in the retinal microvasculature and also showed significant iBRB breakdown (P < .005). These diabetics had higher VEGF mRNA and protein expression in comparison to controls (P < .01). PM-treated diabetics had normal iBRB function and significantly reduced diabetes-mediated VEGF expression. Diabetic retinal vessels showed disrupted TJ integrity when compared to controls, while PM-treated diabetics demonstrated near-normal configuration. Gal-3(-/-) mice showed significantly less diabetes-mediated iBRB dysfunction, junctional disruption, and VEGF expression changes than their WT counterparts. The data suggests an AGE-mediated disruption of iBRB via upregulation of VEGF in the diabetic retina, possibly modulating disruption of TJ integrity, even after acute diabetes. Prevention of AGE formation or genetic deletion of Gal-3 can effectively prevent these acute diabetic retinopathy changes.

Investigating the effects of physiological bile acids on GLP-1 secretion and glucose tolerance in normal and GLP-1R(-/-) mice

Physiological secretion of bile acids has previously been linked to the regulation of blood glucose. GLP-1 is an intestinal peptide hormone with important glucose-lowering actions, such as stimulation of insulin secretion and inhibition of glucagon secretion. In this investigation, we assessed the ability of several bile acid compounds to secrete GLP-1 in vitro in STC-1 cells. Bile acids stimulated GLP-1 secretion from 3.3- to 6.2-fold but some were associated with cytolytic effects. Glycocholic and taurocholic acids were selected for in vivo studies in normal and GLP-1R(-/-) mice. Oral glucose tolerance tests revealed that glycocholic acid did not affect glucose excursions. However, taurocholic acid reduced glucose excursions by 40% in normal mice and by 27% in GLP-1R(-/-) mice, and plasma GLP-1 concentrations were significantly elevated 30 min post-gavage. Additional studies used incretin receptor antagonists to probe involvement of GLP-1 and GIP in taurocholic acid-induced glucose lowering. The findings suggest that bile acids partially aid glucose regulation by physiologically enhancing nutrient-induced GLP-1 secretion. However, GLP-1 secretion appears to be only part of the glucose-lowering mechanism and our studies indicate that the other major incretin GIP is not involved.

Susceptibility of streptozotocin-induced diabetic rat retinal function and ocular blood flow to acute intraocular pressure challenge.

PURPOSE: To consider whether STZ-induced hyperglycemia renders rat retinal function and ocular blood flow more susceptible to acute intraocular pressure (IOP) challenge.

METHODS: Retinal function (electroretinogram, ERG) was measured during acute IOP challenge (10-100 mmHg, 5 mmHg increments, 3 min/step, vitreal cannulation) in adult Long-Evans rats (6-week old, citrate: n=6, STZ: n=10) 4 weeks after citrate buffer or streptozotocin (STZ, 65 mg/kg, blood glucose > 15 mmol/l) injection. At each IOP, dim and bright flash (-4.56, -1.72 log cd.s.m^-2) ERG responses were recorded to measure inner retinal and ON-bipolar cell function, respectively. Ocular blood flow (laser Doppler flowmetry, citrate; n=6, STZ; n=10) was also measured during acute IOP challenge. Retinae were isolated for qPCR analysis of nitric oxide synthase mRNA expression endothelial, eNos; inducible, iNos; neuronal, nNos).

RESULTS: STZ-induced diabetes increased the susceptibility of inner retinal (IOP at 50% response, 60.1, CI: 57.0-62.0 mmHg vs. citrate: 67.5, CI: 62.1-72.4 mmHg) and ON-bipolar cell function (STZ: 60.3, CI: 58.0-62.8 mmHg vs. citrate: 65.1, CI: 58.0-62.78 mmHg) and ocular blood flow (43.9, CI: 40.8-46.8 vs. citrate: 53.4, CI: 50.7-56.1 mmHg) to IOP challenge. Citrate eyes showed elevated eNos mRNA (+49.7%) after IOP stress, an effect not found in STZ-diabetic eyes (-5.7%, P<0.03). No difference was observed for iNos or nNos (P>0.05) following IOP elevation.

CONCLUSIONS: STZ-induced diabetes increased functional susceptibility during acute IOP challenge. This functional vulnerability is associated with a reduced capacity for diabetic eyes to upregulate eNOS expression and to autoregulate blood flow in response to stress.

Hydrogel-Forming Microneedle Arrays Allow Detection of Drugs and Glucose In Vivo: Potential for Use in Diagnosis and Therapeutic Drug Monitoring

We describe, for the first time the use of hydrogel-forming microneedle (MN) arrays for minimally-invasive extraction and quantification of drug substances and glucose from skin in vitro and in vivo. MN prepared from aqueous blends of hydrolysed poly(methyl-vinylether-co-maleic anhydride) (11.1% w/w) and poly(ethyleneglycol) 10,000 daltons (5.6% w/w) and crosslinked by esterification swelled upon skin insertion by uptake of fluid. Post-removal, theophylline and caffeine were extracted from MN and determined using HPLC, with glucose quantified using a proprietary kit. In vitro studies using excised neonatal porcine skin bathed on the underside by physiologically-relevant analyte concentrations showed rapid (5 min) analyte uptake. For example, mean concentrations of 0.16 μg/mL and 0.85 μg/mL, respectively, were detected for the lowest (5 μg/mL) and highest (35 μg/mL) Franz cell concentrations of theophylline after 5 min insertion. A mean concentration of 0.10 μg/mL was obtained by extraction of MN inserted for 5 min into skin bathed with 5 μg/mL caffeine, while the mean concentration obtained by extraction of MN inserted into skin bathed with 15 μg/mL caffeine was 0.33 μg/mL. The mean detected glucose concentration after 5 min insertion into skin bathed with 4 mmol/L was 19.46 nmol/L. The highest theophylline concentration detected following extraction from a hydrogel-forming MN inserted for 1 h into the skin of a rat dosed orally with 10 mg/kg was of 0.363 μg/mL, whilst a maximum concentration of 0.063 μg/mL was detected following extraction from a MN inserted for 1 h into the skin of a rat dosed with 5 mg/kg theophylline. In human volunteers, the highest mean concentration of caffeine detected using MN was 91.31 μg/mL over the period from 1 to 2 h post-consumption of 100 mg Proplus® tablets. The highest mean blood glucose level was 7.89 nmol/L detected 1 h following ingestion of 75 g of glucose, while the highest mean glucose concentration extracted from MN was 4.29 nmol/L, detected after 3 hours skin insertion in human volunteers. Whilst not directly correlated, concentrations extracted from MN were clearly indicative of trends in blood in both rats and human volunteers. This work strongly illustrates the potential of hydrogel-forming MN in minimally-invasive patient monitoring and diagnosis. Further studies are now ongoing to reduce clinical insertion times and develop mathematical algorithms enabling determination of blood levels directly from MN measurements.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Differences In Metabolic Profile In the Testes of Streptozotocin Induced Diabetes Mellitus Mice As Detected By 1H NMR

Contrary to the traditional view, recent studies suggest that diabetes mellitus has an adverse influence on male reproductive function. Our aim was to determine the affect of diabetes on the testicular environment by identifying and then assessing perturbations in small molecule metabolites. Testes were obtained from control and streptozotocin induced diabetic C57BL/6 mice, two, four and eight weeks post treatment. Diabetic status was confirmed by HbA1c, non fasting blood glucose, physiological condition and body weight. Protein free, low molecular weight, water soluble extracts were assessed using 1H NMR spectroscopy. Principal Component Analysis of the derived profiles was used to classify any variations and specific metabolites were identified based on their spectral pattern. Characteristic metabolite profiles were identified for control and diabetic animals with the most distinctive being from mice with the greatest physical deterioration and loss of bodyweight. Eight streptozotocin treated animals did not develop diabetes and displayed profiles similar to controls. Diabetic mice had decreases in creatine, choline and carnitine and increases in lactate, alanine and myo-inositol. Betaine levels were found to be increased in the majority of diabetic mice but decreased in two animals with severe loss of body weight and physical condition. The association between perturbations in a number of small molecule metabolites known to be influential in sperm function, with diabetic status and physiological condition, adds further impetus to the proposal that diabetes influences important spermatogenic pathways and mechanisms in a subtle and previously unrecognised manner.

Comparison of the anti-diabetic effects of GIP- and GLP-1-receptor activation in obese diabetic (ob/ob) mice: studies with DPP IV resistant N-AcGIP and exendin(1-39)amide

Background The two major incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are being actively explored as anti-diabetic agents because they lower blood glucose through multiple mechanisms. The rapid inactivation of GIP and GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes their biological actions short-lived, but stable agonists such as N-acetylated GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and specific GIP and GLP-1 analogues.

Lys(9) for Glu(9) substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His(7) and Ala(8), can greatly improve resistance to this enzyme. Little research has assessed what effect Glu(9)-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu(9) of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys(9))GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys(9))GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor. (C) 2004 Elsevier Inc. All rights reserved.

Regulation and consequences of differential gene expression in diabetic kidney disease

DIN (diabetic nephropathy) is the leading cause of end-stage renal disease worldwide and develops in 25-40% of patients with Type 1 or Type 2 diabetes mellitus. Elevated blood glucose over long periods together with glomerular hypertension leads to progressive glomerulosclerosis and tubulointerstitial fibrosis in susceptible individuals. Central to the pathology of DIN are cytokines and growth factors such as TGF-beta (transforming growth factor beta) superfamily members, including BMPs (bone morphogenetic protein) and TGF-beta 1, which play key roles in fibrogenic responses of the kidney, including podocyte loss, mesangial cell hypertrophy, matrix accumulation and tubulointerstitial fibrosis. Many of these responses can be mimicked in in vitro models of cells cultured in high glucose. We have applied differential gene expression technologies to identify novel genes expressed in in vitro and in vivo models of DN and, importantly, in human renal tissue. By mining these datasets and probing the regulation of expression and actions of specific molecules, we have identified novel roles for molecules such as Gremlin, IHG-1 (induced in high glucose-1) and CTGF (connective tissue growth factor) in DIN and potential regulators of their bioactions.

A randomised, controlled trial of conventional versus automated weaning from mechanical ventilation using SmartcareTM/PS

Objective
Preliminary assessment of an automated weaning system (SmartCare™/PS) compared to usual management of weaning from mechanical ventilation performed in the absence of formal protocols.


Design and setting
A randomised, controlled pilot study in one Australian intensive care unit.


Patients
A total of 102 patients were equally divided between SmartCare/PS and Control.

Interventions
The automated system titrated pressure support, conducted a spontaneous breathing trial and provided notification of success (“separation potential”).

Measurements and results
The median time from the first identified point of suitability for weaning commencement to the state of “separation potential” using SmartCare/PS was 20 h (interquartile range, IQR, 2–40) compared to 8 h (IQR 2–43) with Control (log-rank P = 0.3). The median time to successful extubation was 43 h (IQR 6–169) using SmartCare/PS and 40 (14–87) with Control (log-rank P = 0.6). Unadjusted, the estimated probability of reaching “separation potential” was 21% lower (95% CI, 48% lower to 20% greater) with SmartCare/PS compared to Control. Adjusted for other covariates (age, gender, APACHE II, SOFAmax, neuromuscular blockade, corticosteroids, coma and elevated blood glucose), these estimates were 31% lower (95% CI, 56% lower to 9% greater) with SmartCare/PS. The study groups showed comparable rates of reintubation, non-invasive ventilation post-extubation, tracheostomy, sedation, neuromuscular blockade and use of corticosteroids.

Conclusions
Substantial reductions in weaning duration previously demonstrated were not confirmed when the SmartCare/PS system was compared to weaning managed by experienced critical care specialty nurses, using a 1:1 nurse-to-patient ratio. The effect of SmartCare/PS may be influenced by the local clinical organisational context.

Recent Advances in Antidiabetic Drug Therapies Targeting the Enteroinsular Axis

The enteroinsular axis (EIA) constitutes a physiological signalling system whereby intestinal endocrine cells secrete incretin hormones following feeding that potentiate insulin secretion and contribute to the regulation of blood glucose homeostasis. The two key hormones responsible are named glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Recent years have witnessed sustained development of antidiabetic therapies that exploit the EIA. Current clinical compounds divide neatly into two classes. One concerns analogues or mimetics of GLP-1, such as exenatide (Byetta) or liraglutide (NN2211). The other group comprises the gliptins (e. g. sitagliptin and vildagliptin) which boost endogenous incretin activity by inhibiting the enzyme dipeptidyl peptidase 4 (DPP 4) that degrades both GLP-1 and GIP. Ongoing research indicates that further incretin and gliptin compounds will become available for clinical use in the near future, offering comparable or improved efficacy. For incretin analogues there is the prospect of prolonged duration of action and alternative routes of administration. This review focuses on recent advances in pre-clinical research and their translation into clinical studies to provide future therapies for type 2 diabetes targeting the EIA.

Metabolic profile changes in the testes of mice with streptozotocin-induced type 1 diabetes mellitus

Contrary to the traditional view, recent studies suggest that diabetes mellitus has an adverse influence on male reproductive function. Our aim was to determine the effect of diabetes on the testicular environment by identifying and then assessing perturbations in small molecule metabolites. Testes were obtained from control and streptozotocin-induced diabetic C57BL/6 mice, 2, 4 and 8 weeks post-treatment. Diabetic status was confirmed by glycated haemoglobin, non-fasting blood glucose, physiological condition and body weight. A novel extraction procedure was utilized to obtain protein free, low-molecular weight, water soluble extracts which were then assessed using H-1 nuclear magnetic resonance spectroscopy. Principal component analysis of the derived profiles was used to classify any variations, and specific metabolites were identified based on their spectral pattern. Characteristic metabolite profiles were identified for control and type 1 diabetic animals with the most distinctive being from mice with the largest physical deterioration and loss of body weight. Eight streptozotocin-treated animals did not develop diabetes and displayed profiles similar to controls. Diabetic mice had decreases in creatine, choline and carnitine and increases in lactate, alanine and myo-inositol. Betaine levels were found to be increased in the majority of diabetic mice but decreased in a few animals with severe loss of body weight and physical condition. The association between perturbations in a number of small molecule metabolites known to be influential in sperm function, with diabetic status and physiological condition, adds further impetus to the proposal that diabetes influences important spermatogenic pathways and mechanisms in a subtle and previously unrecognized manner.