The effect of glucose concentrations in the medium on expression of insulin receptors in human lymphocytes B and T: an in vitro study

Context: Insulin is one of the most-known factors that influence the intensity of cell-bound glucose transport. However, in order to react to this hormone, a cell needs specific receptors present in its membrane. The aim of this work was to investigate the insulin receptor expression in B and T cells under incubation with pathological glucose concentrations, respond hyperglycemia and hypoglycemia. Materials and Methods: Isolated B and T cells were cultivated in different concentrations of glucose (high, low and normal). The expression of insulin receptors was investigated using methods of immunocytochemistry and flow cytometry. Results: Incubation for 24?h of lymphocytes in pathological glucose concentrations seems to only have a slight influence on the expression of insulin receptors. No insulin receptor expression has been found in lymphocytes T incubated in both pathological concentrations of glucose. Different concentrations of glucose in the incubation medium were found to only marginally influence expression of insulin receptors in lymphocytes B. Conclusions: Pathological concentrations of glucose in medium cause a decrease in the percent of cells which show expression of insulin receptors in comparison with normal glucose concentration. Thus, it appears highly probable that the insulin receptors did not arise under pathological glucose concentration in these cells de novo, but in little percent lymphocytes have existed there earlier, before the incubation.     

Rapid murine mixed lymphocyte cultures assessed by emergence of T-cell insulin receptors

Murine T lymphocytes incubated in a mixed lymphocyte culture with allogeneic lymphocytes incompatible for theH-2 region or subregions thereof rapidly develop insulin receptors. In contrast, T cells cultured with syngeneic orH-2- andMlslocus compatible cells do not develop insulin-binding sites. The emergence of insulin receptors is probably an early premitotic event.     

Quantitative flow cytometric analysis of expression of tumor necrosis factor receptor types I and II on mononuclear cells

Abstract

Background: Tumor necrosis factor (TNF)-α is an inflammatory cytokine, the biological effects of which are mediated by the interaction with specific membrane-bound receptors. To assess TNF-α receptor (TNFR) expression, it is important to estimate both the number of cells that carry these receptors and the number of receptors per cell, because the cell fate depends on the balance between TNFRI and TNFRII signaling. Objective: The aim of the present study was to develop an optimized protocol to estimate the level of expression of membrane-bound TNFRI and TNFRII, using QuantiBRITE PE calibration beads. Materials and methods: The percentage of cells that expressed membrane-bound TNFRI and TNFRII and the mean number of receptors per cell were determined by flow cytometry using PE-labeled antibodies against TNFR. To create a calibration curve and convert cell fluorescence intensity values to absolute numbers of receptors, we used QuantiBRITE PE beads. Results: CD19⁺ B lymphocytes had the least percentage of cells expressing TNFRI and the greatest number of receptor molecules per cell, whereas CD3⁺ T lymphocytes had the greatest percentage of cells expressing TNFRII and the lowest density of these receptors. We also established that stimulation of peripheral blood mononuclear cells (PBMCs) with the lipopolysaccharide (LPS) significantly increased the number of TNFRI and TNFRII on CD14⁺ monocytes. Conclusion: Application of the protocol-identified differences in the percentage of cells that expressed TNFRs, as well as the absolute number of receptors per cell, among different subpopulations of PBMCs, and between PBMCs cultured with and without LPS.     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

Properties and Regulation of the T Lymphocyte Insulin Receptor

Abstract

Primary human T lymphocytes that have been mitogen activated in chemically defined medium express cell surface insulin receptors. The receptor is identical to other mammalian insulin receptors in binding properties, including: pH dependency, ligand affinity, hormone specificity, and cooperative interactions. Scatchard plots are curvilinear and a ligand-induced increase in dissociation, the property normally associated with “negative cooperativity”, is kinetically demonstrable. In vitro insulin treatment of the receptor-negative, resting T lymphocyte slightly enhances the degree of insulin binding which emerges following cellular activation. Insulin treatment of receptor-positive lymphoblasts results in insulin receptor “down-regulation”. These findings indicate that T lymphoblast insulin receptor concentrations are not significantly influenced by insulin before their emergence but are dramatically regulated by insulin following their appearance at the cell surface.     

The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3-L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis.

The receptors for insulin and insulin-like growth factor I (IGF-I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF-I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA-synthesis, and expression of proto-oncogene c-fos in 3T3-L1 adipocytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD = 3.6 nM, KI of IGF-I greater than 500 nM; IGF-I receptor, KD = 1.1 nM, KI of insulin = 191 nM). Induction of proto-oncogene c-fos mRNA by insulin and IGF-I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50 of insulin, 3.7; IGF-I, 3.9 nM). Similarly, both insulin and IGF-I increased DNA synthesis (EC50 of insulin, 5.8 nM; IGF-I, 4.0 nM), glucose transport activity (EC50 of insulin, 1.7 nM; IGF-I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3-L1 cells the alpha-subunits of insulin and IGF-I receptors have an equal potential to stimulate a metabolic and a mitogenic response.     

Differential effect of insulin and elevated glucose level on adenosine handling in rat T lymphocytes

Reduced proliferation potential is among other T cell functional defects long known feature of diabetes. However, the mechanism responsible for this impairment is still unknown. Our study was undertaken to investigate the effect of changes in glucose and insulin concentrations on adenosine metabolism, transport and receptor-mediated action in rat T lymphocytes. Presented results indicate that vulnerability of T cells to metabolic stress is determined by insulin but not by glucose concentration. However, glucose and insulin differentially affected the activities of adenosine metabolizing enzymes in resting and proliferating T cells. The Con A-induced proliferation of cultured T lymphocytes did not depended on expression level and functional state of nucleoside transporters. Inhibition of adenosine kinase (AK) with 5-iodotubercidin lowers the proliferation potential of T cells to the level observed for insulin-deprived cells. Moreover, insulin-deprived T lymphocytes but not cells cultured in the presence of insulin released significant quantities of adenosine. Under resting conditions, the cAMP level was fivefold higher in cells deprived of insulin comparing to cells cultured in the presence of insulin. Exposition of insulin-deprived T lymphocytes to specific antagonist (ZM241385) of A2a receptor but not to specific antagonist (Alloxazine) of A2b receptor suppressed cAMP elevation and completely restored the proliferation potential of T cells. Concluding, adenosine released by insulin-deprived T cells due to suppressed AK activity by acting on A2a receptors leads to increases in cAMP level and suppression of T cell proliferation. We assume that this mechanism may significantly contribute to immune impairment observed in diabetes.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Insulin and IGF-1 Receptors in A Human Intestinal Adenocarcinoma Cell Line (Caco-2): Regulation of Na+ Glucose Transport Across the Brush Border

Abstract

Both insulin and IGF–1 receptors are present in intestinal mucosal cells, although their role in this tissue is unclear. We have characterized these receptors in a human adenocarcinoma cell line, Caco-2, and examined their role in the regulation of glucose transport and absorption in these cells. The Caco-2 cells demonstrated specific insulin and IGF-1 receptors. They also bound cytochalasin B, suggesting the presence of a glucose transporter-like protein. When grown on membranes, the Caco-2 cells formed columnar, bipolar cells with tight junctions. The monolayer selectively transported D-glucose, and methyl-D-glucose, with complete exclusion of L-glucose, D-mannitol and inulin. The absorption of glucose across the monolayer occurred via a Na⁺/glucose cotransporter, as indicated by a change in short circuit current after addition of glucose to the apical membrane. When examined under several conditions, neither insulin nor IGF-1 had an affect on the transport of glucose across the Caco-2 monolayer, nor the production of lactate by the cells. It is concluded that the insulin and IGF-1 receptors of Caco-2 cells do not regulate glucose transport.     

Receptor-binding kinetics of A-14 and A-19 125I-labelled insulin

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 ¹²⁵I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.     

In vivo and in vitro regulation of IgE production in murine hybridomas

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.     

De novo emergence of insulin-stimulated glucose uptake in human aortic endothelial cells incubated with high glucose

Elevated glucose concentrations have profound effects on cell function. We hypothesized that incubation of human aortic endothelial cells (HAEC) with high glucose increases insulin signaling and develops the appearance of insulin-stimulated glucose uptake by the cells. Compared with 5 mM glucose, incubation of HAEC with 30 mM glucose for up to 48 h increased in a time-dependent manner expression of insulin receptor, insulin receptor substrate (IRS)-1, IRS-2, and GLUT1 proteins. High glucose also increased the specific binding of (125)I-labeled insulin in HAEC accompanied by accelerated production of interleukin (IL)-6 and IL-8. Short-term stimulation by 50 microU/ml insulin did not activate [(14)C]glucose uptake by HAEC incubated in 5 mM glucose. However, an addition of insulin to high glucose-exposed endothelial cells led to a significant increase in [(14)C]glucose uptake in a glucose concentration- and time-dependent fashion, reaching a plateau at 48 h of incubation. Furthermore, incubation of HAEC with 30 mM glucose resulted in a new insulin-stimulated extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase phosphorylation and increased lipid peroxidation and production of reactive oxygen species. These studies show for the first time that high glucose increases expression of insulin receptors and downstream elements of the insulin-signaling pathway and transforms "insulin-resistant" aortic endothelial cells into "insulin-sensitive" tissue regarding glucose uptake.     

Glucose response to an oral glucose tolerance test predicts weight change in non-diabetic subjects

Objective: Glucose exerts a dual action in the regulation of energy balance, consisting of inhibition of energy intake and stimulation of energy expenditure. Whether blood glucose affects long‐term regulation of body weight in humans remains to be established. We sought to test the hypothesis that the post‐challenge glucose response is a predictor of weight change. Research Methods and Procedures: We performed a prospective analysis of the impact of glucose response to an oral glucose tolerance test (OGTT) and a mixed‐meal test (MT) on subsequent changes in body weight (BW) on 253 Pima Indians (166 men and 87 women) with normal glucose regulation at baseline and follow‐up (follow‐up: 7 ± 4 years). Main outcome measures included BW change (total, percent, and annual), plasma glucose and insulin concentrations during OGTT and MT [total and incremental areas under the curve (AUCs)], resting metabolic rate (RMR; indirect calorimetry), and insulin action (euglycemic‐hyperinsulinemic clamp). Results: Total and incremental glucose AUCs during the OGTT (but not the MT) were negatively associated with BW change (total, percent, and annual), both before and after adjusting for sex, age, initial BW, follow‐up time, insulin action, RMR, fasting plasma glucose and insulin concentrations, and insulin response. Total and incremental glucose AUCs during the OGTT were independent determinants of final BW with age, initial BW, follow‐up time, fasting plasma insulin concentrations, and RMR. Discussion: Higher post‐challenge glucose response protects against BW gain in subjects with normal glucose regulation. We propose that this action may be because of the effect of glucose on food intake and/or thermogenesis.     

Increased Insulin Secretion by Muscarinic M1 and M3 Receptor Function from Rat Pancreatic Islets in vitro

Parasympathetic system plays an important role in insulin secretion from the pancreas. Cholinergic effect on pancreatic beta cells exerts primarily through muscarinic receptors. In the present study we investigated the specific role of muscarinic M1 and M3 receptors in glucose induced insulin secretion from rat pancreatic islets in vitro. The involvement of muscarinic receptors was studied using the antagonist atropine. The role of muscarinic M1 and M3 receptor subtypes was studied using subtype specific antagonists. Acetylcholine agonist, carbachol, stimulated glucose induced insulin secretion at low concentrations (10⁻⁸–10⁻⁵ M) with a maximum stimulation at 10⁻⁷ M concentration. Carbachol-stimulated insulin secretion was inhibited by atropine confirming the role of muscarinic receptors in cholinergic induced insulin secretion. Both M1 and M3 receptor antagonists blocked insulin secretion induced by carbachol. The results show that M3 receptors are functionally more prominent at 20 mM glucose concentration when compared to M1 receptors. Our studies suggest that muscarinic M1 and M3 receptors function differentially regulate glucose induced insulin secretion, which has clinical significance in glucose homeostasis.     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes

Objectives

Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.

Methods

Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.

Results

The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of?~?63 and?~?72 kDa confirmed in western blotting, respectively.

Conclusion

The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

     

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI₂ levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI₂, in T1DM mice.     

Integrin expression and H2O2 production in circulating and splenic leukocytes of obese rats

Objective: Obesity is associated with oxidative stress and inflammation. We hypothesized that the pro‐inflammatory state in obesity may result in spontaneous activation and, hence, increased generation of reactive oxygen species (ROS) and integrin expression in the circulating leukocytes. Methods: Flow cytometry was used to determine integrin expression (immunostaining) as well as superoxide and hydrogen peroxide productions (fluorescent probes) in the peripheral blood and splenic leukocyte of 24‐week‐old male obese normotensive and not‐as‐yet diabetic Zucker rats (n = 6) and their lean counterparts (n = 6). Results: Obese rats had hyperlipidemia and normal arterial pressure, plasma glucose, and creatinine concentrations. Nevertheless, obese rats exhibited increased hydrogen peroxide production by circulating and splenic CD4+ and CD8+ T lymphocytes and by splenic macrophages. This was accompanied by up‐regulations of CD11a expression in the peripheral blood and splenic CD4+ T cells, CD11b in circulating macrophages, and CD11a and CD18 in circulating granulocytes. Conclusion: The study revealed direct evidence of spontaneous leukocyte activation and increased ROS generation by T lymphocytes and monocytes in the peripheral blood of obese Zucker rats before the development of diabetes or hypertension. These findings illustrate the link between obesity, oxidative stress, and inflammation.     

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.