A 338-bp proximal fragment of the glucose transporter type 2 (GLUT2) promoter drives reporter gene expression in the pancreatic islets of transgenic mice.

The high Km glucose transporter GLUT2 is a membrane protein expressed in tissues involved in maintaining glucose homeostasis, and in cells where glucose-sensing is necessary. In many experimental models of diabetes, GLUT2 gene expression is decreased in pancreatic beta-cells, which could lead to a loss of glucose-induced insulin secretion. In order to identify factors involved in pancreatic beta-cell specific expression of GLUT2, we have recently cloned the murine GLUT2 promoter and identified cis-elements within the 338-bp of the proximal promoter capable of binding islet-specific trans-acting factors. Furthermore, in transient transfection studies, this 338-bp fragment could efficiently drive the expression of the chloramphenicol acetyl transferase (CAT) gene in cell lines derived from the endocrine pancreas, but displayed no promoter activity in non-pancreatic cells. In this report, we tested the cell-specific expression of a CAT reporter gene driven by a short (338 bp) and a larger (1311 bp) fragment of the GLUT2 promoter in transgenic mice. We generated ten transgenic lines that integrated one of the constructs. CAT mRNA expression in transgenic tissues was assessed using the RNAse protection assay and the quantitative reverse transcribed polymerase chain reaction (RT-PCR). Overall CAT mRNA expression for both constructs was low compared to endogenous GLUT2 mRNA levels but the reporter transcript could be detected in all animals in the pancreatic islets and the liver, and in a few transgenic lines in the kidney and the small intestine. The CAT protein was also present in Langerhans islets and in the liver for both constructs by immunocytochemistry. These findings suggest that the proximal 338 bp of the murine GLUT2 promoter contain cis-elements required for the islet-specific expression of GLUT2.

P-selectin glycoprotein ligand-1 decameric repeats regulate selectin-dependent rolling under flow conditions.

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins to support leukocyte rolling along vascular wall. L- and P-selectin bind to N-terminal tyrosine sulfate residues and to core-2 O-glycans attached to Thr-57, whereas tyrosine sulfation is not required for E-selectin binding. PSGL-1 extracellular domain contains decameric repeats, which extend L- and P-selectin binding sites far above the plasma membrane. We hypothesized that decamers may play a role in regulating PSGL-1 interactions with selectins. Chinese hamster ovary cells expressing wild-type PSGL-1 or PSGL-1 molecules exhibiting deletion or substitution of decamers with the tandem repeats of platelet glycoprotein Ibalpha were compared in their ability to roll on selectins and to bind soluble L- or P-selectin. Deletion of decamers abrogated soluble L-selectin binding and cell rolling on L-selectin, whereas their substitution partially reversed these diminutions. P-selectin-dependent interactions with PSGL-1 were less affected by decamer deletion. Videomicroscopy analysis showed that decamers are required to stabilize L-selectin-dependent rolling. Importantly, adhesion assays performed on recombinant decamers demonstrated that they directly bind to E-selectin and promote slow rolling. Our results indicate that the role of decamers is to extend PSGL-1 N terminus far above the cell surface to support and stabilize leukocyte rolling on L- or P-selectin. In addition, they function as a cell adhesion receptor, which supports approximately 80% of E-selectin-dependent rolling.

New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes.

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.

Comparison of [3H]tamsulosin and [3H]prazosin binding to wild-type and constitutively active alpha1B-adrenoceptors.

We have tested the hypothesis that smaller alpha1B-adrenoceptor labeling by [3H]tamsulosin compared to [3H]prazosin is related to differential recognition of agonist low affinity states. Paired saturation binding experiments with [3H]prazosin and [3H]tamsulosin were performed in membrane preparations from rat liver and Rat- fibroblasts stably transfected with wild-type hamster alpha1B-adrenoceptors or a constitutively active mutant thereof. In all three settings [3H]tamsulosin labeled significantly fewer alpha1B-adrenoceptors than [3H]prazosin. In noradrenaline competition binding experiments, the percentage of agonist low affinity sites was smallest for the constitutively active alpha1B-adrenoceptor but the percentage of agonist low affinity sites recognized by [3H]tamsulosin and [3H]prazosin did not differ significantly. We conclude that [3H]tamsulosin labels fewer alpha1B-adrenoceptors than [3H]prazosin but this is not fully explained by a poorer labeling of agonist low affinity sites.

Alcohol consumption and incidence of type 2 diabetes. Results from the CoLaus study.

BACKGROUND AND AIMS: Moderate alcohol consumption has been shown to decrease the risk of type 2 diabetes (T2DM), but whether this association is also valid for impaired fasting glucose (IFG) is less well known. We aimed at assessing the impact of alcohol consumption and of type of alcoholic beverage on the incidence of T2DM and T2DM + IFG. METHODS AND RESULTS: As many as 4765 participants (2613 women, mean age 51.7 ± 10.5 years) without T2DM at baseline and followed for an average of 5.5 years. The association between alcohol consumption, type of alcoholic beverage and outcomes was assessed after adjustment for a validated T2DM risk score. During follow-up 284 participants developed T2DM and 643 developed IFG. On bivariate analysis, alcohol consumption was positively associated with the risk of developing T2DM or T2DM + IFG. Moderate (14-27 units/week) alcohol consumption tended to be associated with a lower risk of T2DM, but no protective effect was found for T2DM + IFG. Multivariable-adjusted odds ratio (OR) and (95% confidence interval) for T2DM: 0.89 (0.65-1.22), 0.66 (0.42-1.03) and 1.63 (0.93-2.84) for 1-13, 14-27 and 28 + units/week, respectively (p for quadratic trend < 0.005). For T2DM + IFG, the corresponding ORs were 1.09 (0.90-1.32), 1.33 (1.02-1.74) and 1.54 (0.99-2.39), respectively, p for trend = 0.03. No specific effect of alcoholic beverage (wine, beer or spirits) was found for T2DM or for T2DM + IFG. CONCLUSION: Moderate alcohol consumption is associated with a reduced risk of developing T2DM, but not of developing T2DM + IFG. No specific effect of type of alcoholic beverage was found.

Post-axial polydactyly type A2, overgrowth and autistic traits associated with a chromosome 13q31.3 microduplication encompassing miR-17-92 and GPC5.

Genomic rearrangements at chromosome 13q31.3q32.1 have been associated with digital anomalies, dysmorphic features, and variable degree of mental disability. Microdeletions leading to haploinsufficiency of miR17∼92, a cluster of micro RNA genes closely linked to GPC5 in both mouse and human genomes, has recently been associated with digital anomalies in the Feingold like syndrome. Here, we report on a boy with familial dominant post-axial polydactyly (PAP) type A, overgrowth, significant facial dysmorphisms and autistic traits who carries the smallest germline microduplication known so far in that region. The microduplication encompasses the whole miR17∼92 cluster and the first 5 exons of GPC5. This report supports the newly recognized role of miR17∼92 gene dosage in digital developmental anomalies, and suggests a possible role of GPC5 in growth regulation and in cognitive development.

Type of prey influences biology and consumption rate of Orius insidiosus (Say) (Hemiptera, Anthocoridae)

Generalist predators are capable of consuming different types of prey, and as each prey may have distinct nutritional values, each may have a distinct impact on the biology of the predator. Therefore, the aim of this study was to determine how the consumption of different prey influences certain biological characteristics and the predatory capacity of Orius insidiosus (Say). The investigation was performed in climatic chamber at 25 ±1 ºC, RH 70 ± 10% and fotophase 12. Eggs of Anagasta kuehniella (Zeller), adults of Caliothrips phaseoli (Hood) and nymphs of Aphis gossypii Glover were used as prey and were provided daily ad libitum for all the mobile stages of the predator. The results showed that biological parameters of O. insidiosus are affected differently depending on the type of prey ingested. The development time of the nymphal stage was 13.1, 11.23 and 10.25 days for O. insidiosus feeding on eggs of A. kuehniella, nymphs of A. gossypii and adults of C. phaseoli, respectively. Longevity was five times larger for adults fed on eggs of A. kuehniella (56.25 days) compared to that of adults that preyed on nymphs of A. gossypii (11.44 days), and four times larger when the prey were adults of C. phaseoli (13.58 days). The consumption of eggs of A. kuehniella by predator females resulted in a shorter pre-oviposition period (3.2 days) and a longer oviposition period (44.4 days) when compared to the consumption of other types of prey. In addition, fecundity was increased with the consumption of eggs of A. kuehniella (195.25 eggs laid / female) when compared to feeding on the other prey, C. phaseoli (70.00 eggs laid / female) or A. gossypii (22.50 eggs laid / female). However, the consumption of aphids was larger (148.28 nymphs/ nymphal stage) than that of thrips (74.10 thrips / nymphal stage) or eggs of A. kuehniella (37.03 eggs /nymphal stage) for all of the nymphal stages of the predator. The results indicate that the eggs of A. kuehniella are the type of prey best suited for rearing this predator insect under laboratory conditions. Also fecundity was better with this prey even though the predator consumed during its nymphal stage a lesser quantity of eggs compared to the other prey.

The sand rat, Psammomys obesus, develops type 2 diabetic retinopathy similar to humans.

PURPOSE: Diabetic retinopathy (DR) is a leading cause of blindness, yet pertinent animal models are uncommon. The sand rat (Psammomys obesus), exhibiting diet-induced metabolic syndrome, might constitute a relevant model. METHODS: Adult P. obesus (n = 39) were maintained in captivity for 4 to 7 months and fed either vegetation-based diets (n = 13) or standard rat chow (n = 26). Although plant-fed animals exhibited uniform body weight and blood glucose levels over time, nearly 60% of rat chow-raised animals developed diabetes-like symptoms (test group). Animals were killed, and their eyes and vitreous were processed for immunochemistry. RESULTS: Compared with plant-fed animals, diabetic animals showed many abnormal vascular features, including vasodilation, tortuosity, and pericyte loss within the blood vessels, hyperproteinemia and elevated ratios of proangiogenic and antiangiogenic growth factors in the vitreous, and blood-retinal barrier breakdown. Furthermore, there were statistically significant decreases in retinal cell layer thicknesses and densities, accompanied by profound alterations in glia (downregulation of glutamine synthetase, glutamate-aspartate transporter, upregulation of glial fibrillar acidic protein) and many neurons (reduced expression of protein kinase Cα and Cξ in bipolar cells, axonal degeneration in ganglion cells). Cone photoreceptors were particularly affected, with reduced expression of short- and mid-/long-wavelength opsins. Hypercaloric diet nondiabetic animals showed intermediate values. CONCLUSIONS: Simple dietary modulation of P. obesus induces a rapid and severe phenotype closely resembling human type 2 DR. This species presents a valuable novel experimental model for probing the neural (especially cone photoreceptor) pathogenic modifications that are difficult to study in humans and for screening therapeutic strategies.

Review of the type material of three species of Polietina Schnabl & Dziedzicki (Diptera, Muscidae)

The type material of three species of Polietina Schnabl & Dziedzicki, 1911 was examined. Polietina flavidicincta (Stein, 1904) is redescribed and a lectotype is designated; P. stellata (Couri, 1982) is considered junior synonym of P. flavithorax (Stein, 1904), and for the latter a lectotype is designated. The neotype previously designated is considered invalid.

Nocturnal hypoglycaemias in type 1 diabetic patients: what can we learn with continuous glucose monitoring?

AIM: In type 1 diabetic patients (T1DM), nocturnal hypoglycaemias (NH) are a serious complication of T1DM treatment; self-monitoring of blood glucose (SMBG) is recommended to detect them. However, the majority of NH remains undetected on an occasional SMBG done during the night. An alternative strategy is the Continuous glucose monitoring (CGMS), which retrospectively shows the glycaemic profile. The aims of this retrospective study were to evaluate the true incidence of NH in T1DM, the best SMBG time to predict NH, the relationship between morning hyperglycaemia and NH (Somogyi phenomenon) and the utility of CGMS to reduce NH. METHODS: Eighty-eight T1DM who underwent a CGMS exam were included. Indications for CGMS evaluation, hypoglycaemias and correlation with morning hyperglycaemias were recorded. The efficiency of CGMS to reduce the suspected NH was evaluated after 6-9 months. RESULTS: The prevalence of NH was 67% (32% of them unsuspected). A measured hypoglycaemia at bedtime (22-24 h) had a sensitivity of 37% to detect NH (OR=2.37, P=0.001), while a single measure < or =4 mmol/l at 3-hour had a sensitivity of 43% (OR=4.60, P<0.001). NH were not associated with morning hyperglycaemias but with morning hypoglycaemias (OR=3.95, P<0.001). After 6-9 months, suspicions of NH decreased from 60 to 14% (P<0.001). CONCLUSION: NH were highly prevalent and often undetected. SMBG at bedtime, which detected hypoglycaemia had sensitivity almost equal to that of 3-hour and should be preferred because it is easier to perform. Somogyi phenomenon was not observed. CGMS is useful to reduce the risk of NH in 75% of patients.

A quantitative analysis of antagonism and inverse agonism at wild-type and constitutively active hamster alpha1B-adrenoceptors.

In order to characterize inverse agonism at alpha1B-adrenoceptors, we have compared the concentration-response relationships of several quinazoline and non-quinazoline alpha1-adrenoceptor antagonists at cloned hamster wild-type (WT) alpha1B-adrenoceptors and a constitutively active mutant (CAM) thereof upon stable expression in Rat-1 fibroblasts. Receptor activation or inhibition thereof was assessed as [3H]inositol phosphate (IP) accumulation. Quinazoline (alfuzosin, doxazosin, prazosin, terazosin) and non-quinazoline alpha1-adrenoceptor antagonists (BE 2254, SB 216,469, tamsulosin) concentration-dependently inhibited phenylephrine-stimulated IP formation at both WT and CAM with Ki values similar to those previously found in radioligand binding studies. At CAM in the absence of phenylephrine, the quinazolines produced concentration-dependent inhibition of basal IP formation; the maximum inhibition was approximately 55%, and the corresponding EC50 values were slightly smaller than the Ki values. In contrast, BE 2254 produced much less inhibition of basal IP formation, SB 216,469 was close to being a neutral antagonist, and tamsulosin even weakly stimulated IP formation. The inhibitory effects of the quinazolines and BE 2254 as well as the stimulatory effect of tamsulosin were equally blocked by SB 216,469 at CAM. At WT in the absence of phenylephrine, tamsulosin did not cause significant stimulation and none of the other compounds caused significant inhibition of basal IP formation. We conclude that alpha1-adrenoceptor antagonsits with a quinazoline structure exhibit greater efficacy as inverse agonists than those without.

Association between the A-2518G polymorphism in the monocyte chemoattractant protein-1 gene and insulin resistance and Type 2 diabetes mellitus.

AIMS/HYPOTHESIS: The molecular mechanisms of obesity-related insulin resistance are incompletely understood. Macrophages accumulate in adipose tissue of obese individuals. In obesity, monocyte chemoattractant protein-1 (MCP-1), a key chemokine in the process of macrophage accumulation, is overexpressed in adipose tissue. MCP-1 is an insulin-responsive gene that continues to respond to exogenous insulin in insulin-resistant adipocytes and mice. MCP-1 decreases insulin-stimulated glucose uptake into adipocytes. The A-2518G polymorphism in the distal regulatory region of MCP-1 may regulate gene expression. The aim of this study was to investigate the impact of this gene polymorphism on insulin resistance. METHODS: We genotyped the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort ( n=3307). Insulin resistance, estimated by homeostasis model assessment, and Type 2 diabetes were diagnosed in 803 and 635 patients respectively. RESULTS: Univariate analysis revealed that plasma MCP-1 levels were significantly and positively correlated with WHR ( p=0.011), insulin resistance ( p=0.0097) and diabetes ( p<0.0001). Presence of the MCP-1 G-2518 allele was associated with decreased plasma MCP-1 ( p=0.017), a decreased prevalence of insulin resistance (odds ratio [OR]=0.82, 95% CI: 0.70-0.97, p=0.021) and a decreased prevalence of diabetes (OR=0.80, 95% CI: 0.67-0.96, p=0.014). In multivariate analysis, the G allele retained statistical significance as a negative predictor of insulin resistance (OR=0.78, 95% CI: 0.65-0.93, p=0.0060) and diabetes (OR=0.80, 95% CI: 0.66-0.96, p=0.018). CONCLUSIONS/INTERPRETATION: In a large cohort of Caucasians, the MCP-1 G-2518 gene variant was significantly and negatively correlated with plasma MCP-1 levels and the prevalence of insulin resistance and Type 2 diabetes. These results add to recent evidence supporting a role for MCP-1 in pathologies associated with hyperinsulinaemia.