SGLT2 inhibitors:glucuretic treatment for type 2 diabetes

Sodium glucose co-transporter-2 (SGLT2) inhibitors offer a novel approach to treat diabetes by reducing hyperglycaemia via increased glucosuria. This approach reduces renal glucose reabsorption in the proximal renal tubules providing an insulin-independent mechanism to lower blood glucose. The glucuretics are advanced in clinical development and dapagliflozin has received most extensive study. Once daily dapaglifolozin as monotherapy or as add-on to metformin for 12-24 weeks in type 2 diabetic patients (baseline HbA 8-9%) reduced HbA by about 0.5-1%, accompanied by weight loss (2-3 kg) and without significant risk of hypoglycaemia. Dapagliflozin has reduced insulin requirement and improved glycaemic control without weight gain in insulin-treated patients. A mild osmotic diuresis associated with glucuretic therapy may account for a small increase in haematocrit (1-2%) and reduced blood pressure (2-5 mmHg). Dehydration and altered electrolyte balance have not been encountered. Urinary tract and genital infections increased in most studies with dapagliflozin, but were typically mild - resolving with selfmedication or standard intervention. Thus glucuretics provide a novel insulin-independent approach for control of hyperglycaemia which does not incur hypoglycaemia, promotes weight loss, may reduce blood pressure and offers compatibility with other glucose-lowering agents. © 2010 The Author(s).

Astrocytic GABA transporter GAT-1 dysfunction in experimental absence seizures

An enhanced tonic GABA-A inhibition in the thalamus plays a crucial role in experimental absence seizures, and has been attributed, on the basis of indirect evidence, to a dysfunction of the astrocytic GABA transporter-1 (GAT-1). Here, the GABA transporter current was directly investigated in thalamic astrocytes from a well-established genetic model of absence seizures, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), and its non-epileptic control (NEC) strain. We also characterized the novel form of GABAergic and glutamatergic astrocyte-to-neuron signalling by recording slow outward currents (SOCs) and slow inward currents (SICs), respectively, in thalamocortical (TC) neurons of both strains. In patch-clamped astrocytes, the GABA transporter current was abolished by combined application of the selective GAT-1 and GAT-3 blocker, NO711 (30µM) and SNAP5114 (60µM), respectively, to GAERS and NEC thalamic slices. NO711 alone significantly reduced (41%) the transporter current in NEC, but had no effect in GAERS. SNAP5114 alone reduced by half the GABA transporter current in NEC, whilst it abolished it in GAERS. SIC properties did not differ between GAERS and NEC TC neurons, whilst moderate changes in SOC amplitude and kinetics were observed. These data provide the first direct demonstration of a malfunction of the astrocytic thalamic GAT-1 transporter in absence epilepsy and support an abnormal astrocytic modulation of thalamic ambient GABA levels. Moreover, while the glutamatergic astrocyte-neuron signalling is unaltered in the GAERS thalamus, the changes in some properties of the GABAergic astrocyte-neuron signaling in this epileptic strain may contribute to the generation of absence seizures.

The Role of Aquaporin 3 (AQP3) in Breast Cancer

The increasing prevalence of breast cancer (BC) in different parts of the world, particularly in the UK, highlights the importance of research into the aetiology and pathology of the disease. BC is the most common malignancy affecting women worldwide. Aquaporins (AQPs) are membrane protein channels that regulate cellular water flow. Recently, studies have demonstrated that expression of AQP3 is up-regulated in cancerous breast tissue. The present study examines the role of AQP3 in BC cell biology. Examination of clinical cases of BC showed higher AQP3 gene and protein expression in cancer tissues compared to healthy border tissues. In distinct clinicopathological groups however there were no differences observed with regards to AQP3 expression, suggesting that AQP3 expression may not be a predictor of lymph node infiltration or tumour grade. shRNA technology was used to knockdown gene expression of AQP3 in the invasive MDA-MB-231 BC cellular model. Cellular proliferation, migration, invasion, adhesion and response to the 5- fluorouracil (5-FU) based chemotherapy treatment were investigated in parental and knockdown cell line. AQP3 knockdown cells showed reduction in cellular proliferation, migration, invasion and increase in cell sensitivity to 5-FU compared with wild type (WT) or scrambled control (SC) cells. The effects of AQP3 knockdown on cellular glycolytic ability and ATP cellular content were quantified. Indirect glucose uptake was also measured by quantifying reconditioned media. AQP3 knockdown cells showed significantly lower levels of glucose uptake as compared to WT or SC. However there was no difference in the glycolytic ability and ATP content of the cells suggesting AQP3 has no role in cancer cell energetics. These data collectively suggest AQP3 expression is associated with the BC disease clinically and plays a role in multiple important aspects of BC pathophysiology, thus AQP3 represents a novel target for therapeutic intervention.

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*. encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Δ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a nonethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Δ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein-2 (UCP-2) impairs β-cell function and UCP-2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP-2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5–22 mmol/l)±leptin (0–10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose-dependently increased UCP-2 expression in all islet batches, maximally by three-fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP-2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP-2 expression in human islets and provides novel evidence of its role in the loss of β-cell function in diabetes.

Effects of lipoic acid and dihydrolipoic acid on total erythrocytic thiols under conditions of restricted glucose in vitro

The effects of lipoic acid and dihydrolipoic acid were explored on total thiol maintenance in diabetic and non-diabetic human erythrocytes in vitro over 22 hr in a 37°C incubation system with no added glucose. Over 18-22.5 hr after treatment in both non-diabetic and diabetic cells, lipoic acid (1 mM) was associated with greater loss of cellular thiols than dihydrolipoic acid (1 mM), compared to respective control values. At 0.1 mM, in non-diabetic cells, although lipoic acid-treated cells' thiol levels were significantly lower than control, there was no significant difference between dihydrolipoic acid-treated cells and control cells regarding thiol levels. In addition, at 0.1 mM, dihydrolipoic acid-treated diabetic cells showed a reduction in thiol levels compared to control. At 0.01 mM, lipoic acid-treated cells had significantly lower measured thiol levels compared with diabetic cells exposed to dihydrolipoic acid, whereas in non-diabetic cells, dihydrolipoic acid-treated erythrocytic thiol levels were significantly greater than those treated with lipoic acid, although there were no other significant differences between the groups. At 22.5 hr, control values of methaemoglobin rose to 6.4 ± 1.1% in diabetic cells and 3.6 ± 2.1% in non-diabetic cells. Lipoic acid (1 mM) showed greater methaemoglobin formation in diabetic rather than non-diabetic cells (13.6 ± 1.5% versus 11.6 ± 1.5%), whereas dihydrolipoic acid-treated diabetic and non-diabetic cells were less potent in methaemoglobin generation (8.5 ± 2.4% and 8.4 ± 1.4%, respectively). These studies suggest that in certain circumstances such as hypoglycaemia, lipoic acid administration may actually be detrimental to cellular oxidant protection status. © 2006 The Authors.

GIP(Lys16PAL) and GIP(LyS37PAL):novel long-acting acylated analogues of glucose-dependent insulinotropic polypeptide with improved antidiabetic potentia

Glucose-dependent insulinotropic polypeptide (GIP) is a physiological insulin releasing peptide. We have developed two novel fatty acid derivatized GIP analogues, which bind to serum albumin and demonstrate enhanced duration of action in vivo. GIP(Lys16PAL) and GIP(Lys37PAL) were resistant to dipeptidyl peptidase IV (DPP IV) degradation. In vitro studies demonstrated that GIP analogues retained their ability to activate the GIP receptor through production of cAMP and to stimulate insulin secretion. Intraperitoneal administration of GIP analogues to obese diabetic (ob/ob) mice significantly decreased the glycemic excursion and elicited increased and prolonged insulin responses compared to native GIP. A protracted glucose-lowering effect was observed 24 h following GIP(Lys37PAL) administration. Once a day injection for 14 days decreased nonfasting glucose, improved glucose tolerance, and enhanced the insulin response to glucose. These data demonstrate that fatty acid derivatized GIP peptides represent a novel class of long-acting stable GIP analogues for therapy of type 2 diabetes. © 2006 American Chemical Society.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala8-substituted analogues of GLP-1, (Abu8)GLP-1 and (Val8)GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu8)GLP-1 and (Val8)GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu8)GLP-1 and (Val8)GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val8)GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu8 )GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val8)GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala8 in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val8)GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Clinical evaluation of a novel test strip technology for blood glucose monitoring:accuracy at hypoglycaemic glucose levels

Aim: To evaluate OneTouch® Verio™ test strip performance at hypoglycaemic blood glucose (BG) levels (<3.9mmol/L [<70mg/dL]) at seven clinical studies. Methods: Trained clinical staff performed duplicate capillary BG monitoring system tests on 700 individuals with type 1 and type 2 diabetes using blood from a single fingerstick lancing. BG reference values were obtained using a YSI 2300 STAT™ Glucose Analyzer. The number and percentage of BG values within ±0.83. mmol/L (±15. mg/dL) and ±0.56. mmol/L (±10. mg/dL) were calculated at BG concentrations of <3.9. mmol/L (<70. mg/dL), <3.3. mmol/L (<60. mg/dL), and <2.8. mmol/L (<50. mg/dL). Results: At BG concentrations <3.9. mmol/L (<70. mg/dL), 674/674 (100%) of meter results were within ±0.83. mmol/L (±15. mg/dL) and 666/674 (98.8%) were within ±0.56. mmol/L (±10. mg/dL) of reference values. At BG concentrations <3.3. mmol/L (<60. mg/dL), and <2.8. mmol/L (<50. mg/dL), 358/358 (100%) and 270/270 (100%) were within ±0.56. mmol/L (±10. mg/dL) of reference values, respectively. Conclusion: In this analysis of data from seven independent studies, OneTouch Verio test strips provide highly accurate results at hypoglycaemic BG levels. © 2012 Elsevier Ireland Ltd.

Hydrothermally stable, conformal, sulfated zirconia monolayer catalysts for glucose conversion to 5-HMF

The grafting and sulfation of zirconia conformal monolayers on SBA-15 to create mesoporous catalysts of tunable solid acid/base character is reported. Conformal zirconia and sulfated zirconia (SZ) materials exhibit both Brönsted and Lewis acidity, with the Brönsted/Lewis acid ratio increasing with film thickness and sulfate content. Grafted zirconia films also exhibit amphoteric character, whose Brönsted/Lewis acid site ratio increases with sulfate loading at the expense of base sites. Bilayer ZrO2/SBA-15 affords an ordered mesoporous material with a high acid site loading upon sulfation and excellent hydrothermal stability. Catalytic performance of SZ/SBA-15 was explored in the aqueous phase conversion of glucose to 5-HMF, delivering a 3-fold enhancement in 5-HMF productivity over nonporous SZ counterparts. The coexistence of accessible solid basic/Lewis acid and Brönsted acid sites in grafted SZ/SBA-15 promotes the respective isomerization of glucose to fructose and dehydration of reactively formed fructose to the desired 5-HMF platform chemical.

Extra-nuclear telomerase reverse transcriptase (TERT) regulates glucose transport in skeletal muscle cells

Telomerase reverse transcriptase (TERT) is a key component of the telomerase complex. By lengthening telomeres in DNA strands, TERT increases senescent cell lifespan. Mice that lack TERT age much faster and exhibit age-related conditions such as osteoporosis, diabetes and neurodegeneration. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. We investigated the role of TERT, in regulating cellular glucose utilisation by using the myoblastoma cell line C2C12, as well as primary mouse and human skeletal muscle cells. Inhibition of TERT expression or activity by using siRNA (100. nM) or specific inhibitors (100. nM) reduced basal 2-deoxyglucose uptake by ~. 50%, in all cell types, without altering insulin responsiveness. In contrast, TERT over-expression increased glucose uptake by 3.25-fold. In C2C12 cells TERT protein was mostly localised intracellularly and stimulation of cells with insulin induced translocation to the plasma membrane. Furthermore, co-immunoprecipitation experiments in C2C12 cells showed that TERT was constitutively associated with glucose transporters (GLUTs) 1, 4 and 12 via an insulin insensitive interaction that also did not require intact PI3-K and mTOR pathways. Collectively, these findings identified a novel extra-nuclear function of TERT that regulates an insulin-insensitive pathway involved in glucose uptake in human and mouse skeletal muscle cells. © 2014 Elsevier B.V.

Ghrelin suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans.

OBJECTIVE: The orexigenic gut hormone ghrelin and its receptor are present in pancreatic islets. Although ghrelin reduces insulin secretion in rodents, its effect on insulin secretion in humans has not been established. The goal of this study was to test the hypothesis that circulating ghrelin suppresses glucose-stimulated insulin secretion in healthy subjects. RESEARCH DESIGN AND METHODS: Ghrelin (0.3, 0.9 and 1.5 nmol/kg/h) or saline was infused for more than 65 min in 12 healthy patients (8 male/4 female) on 4 separate occasions in a counterbalanced fashion. An intravenous glucose tolerance test was performed during steady state plasma ghrelin levels. The acute insulin response to intravenous glucose (AIRg) was calculated from plasma insulin concentrations between 2 and 10 min after the glucose bolus. Intravenous glucose tolerance was measured as the glucose disappearance constant (Kg) from 10 to 30 min. RESULTS: The three ghrelin infusions raised plasma total ghrelin concentrations to 4-, 15-, and 23-fold above the fasting level, respectively. Ghrelin infusion did not alter fasting plasma insulin or glucose, but compared with saline, the 0.3, 0.9, and 1.5 nmol/kg/h doses decreased AIRg (2,152 +/- 448 vs. 1,478 +/- 2,889, 1,419 +/- 275, and 1,120 +/- 174 pmol/l) and Kg (0.3 and 1.5 nmol/kg/h doses only) significantly (P < 0.05 for all). Ghrelin infusion raised plasma growth hormone and serum cortisol concentrations significantly (P < 0.001 for both), but had no effect on glucagon, epinephrine, or norepinephrine levels (P = 0.44, 0.74, and 0.48, respectively). CONCLUSIONS: This is a robust proof-of-concept study showing that exogenous ghrelin reduces glucose-stimulated insulin secretion and glucose disappearance in healthy humans. Our findings raise the possibility that endogenous ghrelin has a role in physiologic insulin secretion, and that ghrelin antagonists could improve beta-cell function.

Tab. 1+2+3: Selected principal soil properties in the oil exploitation region of KomiArcticOil (Usinsk) in the Russian tundra

Oil polluted and not oil polluted soils (crude oil hydrocarbons contents: 20-92500 mg/kg dry soil mass) under natural grass and forest vegetation and in a bog in the Russian tundra were compared in their principal soil ecological parameters, the oil content and the microbial indicators. CFE biomass-C, dehydrogenase and arylsulfatase activity were enhanced with the occurrence of crude oil. Using these parameters for purposes of controlling remediation and recultivation success it is not possible to distinguish bctween promotion of microbial activity by oil carbon or soil organic carbon (SOC). For this reason we think that these parameters are not appropriate to indicate a soil damage by an oil impact. In contrast the metabolie quotient (qC02), calculated as the ratio between soil basal respiration and the SIR biomass-C was adequate to indicate a high crude oil contamination in soil. Also, the ß-glucosidase activity (parameter ß-GL/SOC) was correlated negatively with oil in soil. The indication of a soil damage by using the stress parameter qCO, or the specific enzyme activities (activity/SOC) minimizes the promotion effect of the recent SOC content on microbial parameters. Both biomass methods (SIR, CFE) have technical problems in application for crude oil-contaminated and subarctic soils. CFE does not reflect the low C_mic level of the cold tundra soils. We recommend to test every method for its suitability before any data collection in series as well as application for cold soils and the application of ecophysiological ratios as R_mic/C_mic, C_mic/SOC or enzymatic activity/SOC instead of absolute data.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.Leukemia advance online publication, 17 June 2016; doi:10.1038/leu.2016.149.