Characterization of the Aquaporin-9 Inhibitor RG100204 In Vitro and in db/db Mice.

Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10-8 M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.

CCR8 signaling influences Toll-like receptor 4 responses in human macrophages in inflammatory diseases.

CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients.

Measurement of MR signal and T2* in lung to characterize a tight skin mouse model of emphysema using single-point imaging.

PURPOSE: To evaluate whether MRI signal and T2* measurements of lung tissue acquired at ultrashort detection times (tds) can detect emphysematous changes in lungs. MATERIALS AND METHODS: MR signal intensity of in vivo mouse lungs was measured at 4.7 T at tds of 0.2 and 0.4 msec using single-point imaging (SPI). T2* was calculated from the measurements obtained at the two tds. Two groups of 8- and 30-week-old Tight Skin (TS) and aged-matched CB57BL/6 mice were examined. The TS mice spontaneously developed emphysema-like alveolar enlargement. In vivo micro-computed tomography (microCT) scanning and histology were used as reference methods. RESULTS: MR signal and T2* were significantly lower in the lungs of TS mice than in controls. There were no significant differences between the different age groups. MR signal in lung parenchyma correlated linearly (P < 0.0001, r = 0.89) with microCT mass density, and T2* correlated linearly (P < 0.0001, r = -0.91) with the alveoli size (mean linear intercept [MLI]). CONCLUSION: The MR signal intensity and T2* measured at short tds can be used as imaging biomarkers to characterize parenchyma density and alveolar size, respectively.

Aged transgenic mice with increased glucocorticoid sensitivity in pancreatic beta-cells develop diabetes.

Glucocorticoids are diabetogenic hormones because they decrease glucose uptake, increase hepatic glucose production, and inhibit insulin release. To study the long-term effects of increased glucocorticoid sensitivity in beta-cells, we studied transgenic mice overexpressing the rat glucocorticoid receptor targeted to the beta-cells using the rat insulin I promoter. Here we report that these mice developed hyperglycemia both in the fed and the overnight-fasted states at 12-15 months of age. Progression from impaired glucose tolerance, previously observed in the same colony at the age of 3 months, to manifest diabetes was not associated with morphological changes or increased apoptosis in the beta-cells. Instead, our current results suggest that the development of diabetes is due to augmented inhibition of insulin secretion through alpha(2)-adrenergic receptors (alpha(2)-ARs). Thus, we found a significantly higher density of alpha(2)-ARs in the islets of transgenic mice compared with controls, based on binding studies with the alpha(2)-AR agonist UK 14304. Furthermore, incubation of islets with benextramine, a selective antagonist of the alpha(2)-AR, restored insulin secretion in response to glucose in isolated islets from transgenic mice, whereas it had no effect on control islets. These results indicate that the chronic enhancement of glucocorticoid signaling in pancreatic beta-cells results in hyperglycemia and impaired glucose tolerance. This effect may involve signaling pathways that participate in the regulation of insulin secretion via the alpha(2)-AR.

Local growth factors are beneficial for the autonomic reinnervation of transplanted islets in rats.

INTRODUCTION: Transplanted islets, being avascular and denervated, receive blood vessels and nerves from the recipient. Reinnervation may account in part for the normalization of islet function in islet transplants. Whether reinnervation is possible to augment is not known. AIMS AND METHODOLOGY: To explore whether reinnervation of transplanted islets is augmented by local addition of growth factors to the graft, syngeneic islets were transplanted to the pancreas of streptozotocin-diabetic Lewis rats with or without pellets locally releasing nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), alone or in combination. The pellets released growth factors for 14 days at a rate of 20 ng/day. After 7 weeks, pancreatic tissue was processed for immunofluorescence of insulin and the neural markers neuropeptide Y (NPY) and tyrosine hydroxylase (TH). RESULTS: Islets were larger and more numerous after treatment with NGF (p = 0.024) and with NGF in combination with VEGF (p = 0.044). Similarly, immunostaining for TH and the C-terminal flanking peptide of NPY (C-PON) was more pronounced after treatment with NGF in combination with VEGF than in controls (both p < 0.05). CONCLUSION: Local growth factor treatment has a beneficial effect on autonomic reinnervation as well as islet integrity and survival of the graft after islet transplantation in rats.

Dose-dependent inhibition by ghrelin of insulin secretion in the mouse.

Ghrelin is produced by stomach oxyntic cells and thought to be involved in the regulation of body weight and food intake. We demonstrate here that the peptide inhibits insulin secretion from overnight-incubated mouse islets in the presence of 8.3, 11.1, and 22.2 mmol/liter glucose. Ghrelin was most efficient at 1 nmol/liter and its effect disappeared by raising the dose more than 25 nmol/liter. Also, insulin secretion in the presence of high K(+) concentrations (20 mmol/liter) was inhibited by ghrelin. Furthermore, when administered iv to mice together with glucose (1 g/kg), ghrelin (50 nmol/kg) inhibited both the rapid 1-min insulin response (364 +/- 90 vs. 985 +/- 114 pmol/liter in controls, P < 0.001) and the area under the 50 min curve of insulin concentration (12.6 +/- 1.2 vs. 15.6 +/- 1.2 nmol/liter x 50 min; P = 0.046) without affecting the glucose disposal rate, insulin sensitivity or glucose effectiveness, i.e. glucose disposal independent from any dynamic change in insulin. The insulinostatic effect of ghrelin was inversely related to insulin sensitivity. In contrast, ghrelin had no influence at the lower dose of 5 nmol/kg and only slightly inhibited insulin secretion at the higher dose of 150 nmol/kg. These findings therefore show that ghrelin inhibits glucose-stimulated insulin secretion in the mouse. The effect is dependent on the dose and elicited on distal signaling steps in islet cells. The results suggest that the islet beta-cells are targets for ghrelin.

Altered beta-cell distribution of pdx-1 and GLUT-2 after a short-term challenge with a high-fat diet in C57BL/6J mice.

Mechanisms involved in the islet adaptation to insulin resistance were examined in mice of the C57BL/6J strain challenged with a high-fat (58%) diet for 8 weeks. Basal hyperglycemia commenced after 1 week, whereas hyperinsulinemia evolved after 8 weeks. Glucose elimination after an intravenous glucose challenge (1 g/kg) was significantly delayed after 1, 4, and 8 weeks on the high-fat diet compared with normal-diet-fed mice. This result was associated with unchanged insulin responses. However, glucose-stimulated insulin secretion from isolated islets was increased in a compensatory fashion at all glucose levels over a wide range (3.3-22 mmol/l) after 8 weeks on the high-fat diet, whereas no compensatory hypersecretion of insulin was evident after 1 or 4 weeks, except at 22 mmol/l glucose. Immunohistochemistry revealed that the islet architecture of insulin and glucagon cells remained intact in islets from mice fed a high-fat diet. However, the nuclear translocation of the homeobox transcription factor, pdx-1, and the plasma membrane translocation of GLUT2 were both impaired in high-fat-fed animals after 1 week. In contrast, the expression of the full-length leptin receptor (ObRb) was not affected by high-fat feeding. The study thus shows that 8 weeks are required for the development of a compensatory hypersecretion of insulin after high-fat feeding in mice, and even then the in vivo insulin secretion is insufficient to normalize impaired glucose tolerance. The early-onset islet dysfunction is accompanied by impaired beta-cell trafficking of two factors, pdx-1 and GLUT-2, which are involved in beta-cell proliferation and glucose recognition. The mechanisms compromising this beta-cell trafficking remain to be established.