Impact of siRNA-Mediated Cofilin-1 Knockdown and Obesity Associated Microenvironment on the Motility of Natural Killer Cells.

The anti-tumor capacity of natural killer (NK) cells heavily relies on their ability to migrate towards their target cells. This process is based on dynamic actinrearrangement, so-called actin treadmilling, andis tightly regulated by proteins such as cofilin-1. The aim of the present study was to identify the role of cofilin-1 (CFL-1) in the migratory behavior of NK cells and to investigate a possible impact of an obesity-associated micromilieu on these cells, as it is known that obesity correlates with various impaired NK cell functions. CFL-1 was knocked-down via transfection of NK-92 cells with respective siRNAs. Obesity associated micromilieu was mimicked by incubation of NK-92 cells with adipocyte-conditioned medium from human preadipocyte SGBS cells or leptin. Effects on CFL-1 levels, the degree of phosphorylation to the inactive pCFL-1 as well as NK-92 cell motility were analyzed. Surprisingly, siRNA-mediated CFL-1 knockdown led to a significant increase of migration, as determined by enhanced velocity and accumulated distance of migration. No effect on CFL-1 nor pCFL-1 expression levels, proportion of phosphorylation and cell migratory behavior could be demonstrated under the influence of an obesity-associated microenvironment. In conclusion, the results indicate a significant effect of a CFL-1 knockdown on NK cell motility.

Influence of Cannabinoid Receptor Deficiency on Parameters Involved in Blood Glucose Regulation in Mice.

Cannabinoids are known to influence hormone secretion of pancreatic islets via G protein­coupled cannabinoid receptor type 1 and 2 (CB1 and CB2). The present study was designed to further investigate the impact of cannabinoid receptors on the parameters involved in insulin secretion and blood glucose recognition. To this end, CB1 and CB2 receptor knockout mice (10-12 week old, both sexes) were characterised at basal state and compared to wild-type mice. The elimination of cannabinoid receptor signalling resulted in alterations of blood glucose concentrations, body weights and insulin levels. Changes were dependent on the deleted receptor type and on the sex. Analyses at mRNA and protein levels provided evidence for the impact of cannabinoid receptor deficiency on the glucose sensing apparatus in the pancreas. Both receptor knockout mouse lines showed decreased mRNA and protein amounts of glucose transporters Glut1 and Glut2, combined with alterations in immunostaining. In addition, pancreatic glucokinase expression was elevated and immunohistochemical labelling was modified in the pancreatic islets. Taken together, CB1 and CB2 signalling pathways seem to influence glucose sensing in ß-cells by affecting glucose transporters and glucokinase. These alterations were more pronounced in CB2 knockout mice, resulting in higher blood glucose and lower plasma insulin levels.

Endocannabinoid System in Hepatic Glucose Metabolism, Fatty Liver Disease, and Cirrhosis.

There is growing evidence that glucose metabolism in the liver is in part under the control of the endocannabinoid system (ECS) which is also supported by its presence in this organ. The ECS consists of its cannabinoid receptors (CBRs) and enzymes that are responsible for endocannabinoid production and metabolism. ECS is known to be differentially influenced by the hepatic glucose metabolism and insulin resistance, e.g., cannabinoid receptor type 1(CB1) antagonist can improve the glucose tolerance and insulin resistance. Interestingly, our own study shows that expression patterns of CBRs are influenced by the light/dark cycle, which is of significant physiological and clinical interest. The ECS system is highly upregulated during chronic liver disease and a growing number of studies suggest a mechanistic and therapeutic impact of ECS on the development of liver fibrosis, especially putting its receptors into focus. An opposing effect of the CBRs was exerted via the CB1 or CB2 receptor stimulation. An activation of CB1 promoted fibrogenesis, while CB2 activation improved antifibrogenic responses. However, underlying mechanisms are not yet clear. In the context of liver diseases, the ECS is considered as a possible mediator, which seems to be involved in the synthesis of fibrotic tissue, increase of intrahepatic vascular resistance and subsequently development of portal hypertension. Portal hypertension is the main event that leads to complications of the disease. The main complication is the development of variceal bleeding and ascites, which have prognostic relevance for the patients. The present review summarizes the current understanding and impact of the ECS on glucose metabolism in the liver, in association with the development of liver cirrhosis and hemodynamics in cirrhosis and its complication, to give perspectives for development of new therapeutic strategies.

Distribution and density of melatonin receptors in human main pancreatic islet cell types.

Recent investigations of our group established that melatonin modulates hormone secretion of pancreatic islets via melatonin receptor types MT1 and MT2. Expression of MT1 and MT2 has been shown in mouse, rat, and human pancreatic islets as well as in the ß-, α-, and δ-cell lines INS-1, αTC1.9, and QGP-1. In view of these earlier investigations, this study was performed to analyze in detail the distribution and density of melatonin receptors on the main islet cell types in human pancreatic tissue obtained from nondiabetic and type 2 diabetic patients. Immunohistochemical analysis established the presence of MT1 and MT2 in ß-, α-, and δ-cells, but notably, with differences in receptor density. In general, the lowest MT1 and MT2 receptor density was measured in α-cells compared to the 2 other cell types. In type 2 diabetic islets, MT1 and MT2 receptor density was increased in δ-cells compared to normoglycemic controls. In human islets in batch culture of a nondiabetic donor, an increase of somatostatin secretion was observed under melatonin treatment while in islets of a type 2 diabetic donor, an inhibitory influence could be observed, especially in the presence of 5.5 mmol/L glucose. These data suggest the following: i) cell-type-specific density of MT1 and MT2 receptors in human pancreatic islets, which should be considered in context of the hormone secretion of islets, ii) the influence of diabetes on density of MT1 and MT2 as well as iii) the differential impact of melatonin on somatostatin secretion of nondiabetic and type 2 diabetic islets.

Daytime-Dependent Changes of Cannabinoid Receptor Type 1 and Type 2 Expression in Rat Liver.

The present study was performed to investigate the diurnal expression pattern of cannabinoid receptor type 1 (CB1) and type 2 (CB2) in liver tissue of 12- and 51-week-old normoglycemic Wistar rats. By using real-time RT-PCR, daytime dependent changes in both age groups and, for both, hepatic Cnr1 and Cnr2 receptor mRNA levels were measured. Highest amount of mRNA was detected in the light period (ZT3, ZT6, and ZT9) while the lowest amount was measured in the dark period (ZT18 and ZT21). Diurnal transcript expression pattern was accompanied by comparable changes of protein level for CB1, as shown by Western blotting. The current results support the conclusion that expression pattern of cannabinoid receptors are influenced by light/dark cycle and therefore seems to be under the control of a diurnal rhythm. These findings might explain the differences in the efficacy of cannabinoid receptor agonists or antagonists. In addition, investigation of liver of streptozotocin (STZ)-treated 12- and 51-week-old rats show alterations in the diurnal profile of both receptors Cnr1 and Cnr2 compared to that of normoglycemic Wistar rats. This suggests an influence of diabetic state on diurnal expression levels of cannabinoid receptors.

Calcium-signaling components in rat insulinoma ß-cells (INS-1) and pancreatic islets are differentially influenced by melatonin.

The pineal secretory product melatonin exerts its influence on the insulin secretion of pancreatic islets by different signaling pathways. The purpose of this study was to analyze the impact of melatonin on calcium-signaling components under different conditions. In a transfected INS-1 cell line overexpressing the human MT2 receptor (hMT2-INS-1), melatonin treatment induced even stronger depressive effects on calcium/calmodulin-dependent kinase 2d and IV (Camk2d, CamkIV) transcripts during 3-isobutyl-1-methylxanthine (IBMX) treatment than in normal INS-1 cells, indicating a crucial influence of melatonin receptor density on transcript-level regulation. In addition, melatonin induced a significant downregulation of calmodulin (Calm1) in IBMX-treated hMT2-INS-1 cells. Long-term administration of melatonin alone reduced CamkIV transcript levels in INS-1 cells; however, transcript levels of Camk2d remained unchanged. The release of insulin was diminished under long-term melatonin treatment. The impact of melatonin also involved reductions in CAMK2D protein during IBMX or forskolin treatments in INS-1 cells, as measured by an enzyme-linked immunosorbent assay, indicating a functional significance of transcriptional changes in pancreatic islets. Furthermore, analysis of melatonin receptor knockout mice showed that the transcript levels of Camk2d, CamkIV, and Calm1 were differentially influenced according to the melatonin receptor subtype deleted. In conclusion, this study provides evidence that melatonin has different impacts on the regulation of Calm1 and Camk. These calcium-signaling components are known as participants in the calcium/calmodulin pathway, which plays an important functional role in the modulation of the ß-cell signaling pathways leading to insulin secretion.

Influence of melatonin receptor signalling on parameters involved in blood glucose regulation.

The pineal hormone melatonin is known to influence insulin secretion via the G-protein-coupled receptor isoforms MT1 and MT2. The present study was aimed to further elucide the impact of melatonin on blood glucose regulation. To this end, mouse lines were used, in which one of the two or both melatonin receptors were deleted. In comparison with wild-type mice of the same age (8-12 months old), increased plasma insulin and melatonin levels and decreased blood glucose levels and body weights were detected in the MT1- and double-knockout lines. The elimination of melatonin receptor signalling also altered blood glucose concentrations, body weight and melatonin and insulin levels when comparing wild-type and receptor knockout mice of different ages (6 wk and 8-12 months old); such changes, however, were dependent on the type of receptor deleted. Furthermore, reverse transcription polymerase chain reaction results provided evidence that melatonin receptor deficiency has an impact on transcript levels of pancreatic islet hormones as well as on pancreatic and hepatic glucose transporters (Glut1 and 2). Under stimulated insulin secretion in the presence of melatonin in the rat insulinoma ß-cells INS-1, the Glut1 transcript level was decreased. In conclusion, the present findings demonstrate that melatonin receptor knockout types affect blood glucose levels, body weight, plasma levels of melatonin and insulin, as well as pancreatic hormone and Glut1 expression in significantly different manners.

Melatonin influences insulin secretion primarily via MT(1) receptors in rat insulinoma cells (INS-1) and mouse pancreatic islets.

Several studies have revealed that melatonin affects the insulin secretion via MT(1) and MT(2) receptor isoforms. Owing to the lack of selective MT(1) receptor antagonists, we used RNA interference technology to generate an MT(1) knockdown in a clonal ß-cell line to evaluate whether melatonin modulates insulin secretion specifically via the MT(1) receptor. Incubation experiments were carried out, and the insulin concentration in supernatants was measured using a radioimmunoassay. Furthermore, the intracellular cAMP was determined using an enzyme-linked immunosorbent assay. Real-time RT-PCR indicated that MT(1) knockdown resulted in a significant increase in the rIns1 mRNA and a significantly elevated basal insulin secretion of INS-1 cells. Incubation with melatonin decreased the amount of glucagon-like peptide 1 or inhibited the glucagon-stimulated insulin release of INS-1 cells, while, in MT(1) -knockdown cells, no melatonin-induced reduction in insulin secretion could be found. No decrease in 3-isobutyl-1-methylxanthine-stimulated intracellular cAMP in rMT(1) -knockdown cells was detectable after treatment with melatonin either, and immunocytochemistry proved that MT(1) knockdown abolished phosphorylation of cAMP-response-element-binding protein. In contrast to the INS-1 cells, preincubation with melatonin did not sensitize the insulin secretion of rMT(1) -knockdown cells. We also monitored insulin secretion from isolated islets of wild-type and melatonin-receptor knockout mice ex vivo. In islets of wild-type mice, melatonin treatment resulted in a decrease in insulin release, whereas melatonin treatment of islets from MT(1) knockout and MT(1/2) double-knockout mice did not show a significant effect. The data indicate that melatonin inhibits insulin secretion, primarily via the MT(1) receptor in rat INS-1 cells and isolated mouse islets.

Phosphorylation of cyclic AMP-response element-binding protein (CREB) is influenced by melatonin treatment in pancreatic rat insulinoma ß-cells (INS-1).

The pineal hormone melatonin exerts its influence on the insulin secretion of pancreatic islets by a variety of signalling pathways. The purpose of the present study was to analyse the impact of melatonin on the phosphorylated transcription factor cAMP-response element-binding protein (pCREB). In pancreatic rat insulinoma ß-cells (INS-1), pCREB immunofluorescence intensities in cell nuclei using digitised confocal image analysis were measured to semi-quantify differences in the pCREB immunoreactivity (pCREB-ir) caused by different treatments. Increasing concentrations of forskolin or 3-isobutyl-1-methylxanthine (IBMX) resulted in a dose-dependent rise of the mean fluorescence intensity in pCREB-ir nuclear staining. Concomitant melatonin application significantly decreased pCREB-ir in INS-1 cells after 30-min, 1-hr and 3-hr treatment. The melatonin receptor antagonists luzindole and 4-phenyl-2-propionamidotetraline (4P-PDOT) completely abolished the pCREB phosphorylation-decreasing effect of melatonin, indicating that both melatonin receptor isoforms (MT(1) and MT(2)) are involved. In a transfected INS-1 cell line expressing the human MT(2) receptor, melatonin caused the greatest reduction in pCREB after IBMX treatment compared with nontransfected INS-1 cells, indicating a crucial influence of melatonin receptor density on pCREB regulation. Furthermore, the downregulation of pCREB by melatonin is concomitantly associated with a statistically significant downregulation of Camk2d transcript levels, as measured after 3 hr. In conclusion, the present study provides evidence that the phosphorylation level of CREB is modulated in pancreatic ß-cells by melatonin. Mediated via CREB, melatonin regulates the expression of genes that play an important functional role in the regulation of ß-cell signalling pathways.

Human and Rodent Cell Lines as Models of Functional Melatonin-Responsive Pancreatic Islet Cells.

Cell culture of different pancreatic islet cell lines, like the murine α-cell line αTC1.9, the rat ß-cell lines INS-1 and INS-1 832/13, and the human δ-cell line QGP-1, can serve as valuable cell models for the analysis of melatonin-dependent modulation of hormone secretion. The paper summarizes in detail the requirements of culture for each cell line and includes batch protocols to stimulate hormone secretion and to treat cells with several melatonin concentrations as previously published. We here describe the processing of collected cell pellets or cell culture supernatants as well as different methods to analyze cell experiments after melatonin treatment on the basis of our own experience. Finally, we outlined for each cell line under which conditions the melatonin treatment should be performed to gain reproducible results.

Melatonin inhibits insulin secretion in rat insulinoma ß-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2.

Melatonin exerts some of its effects via G-protein-coupled membrane receptors. Two membrane receptor isoforms, MT1 and MT2, have been described. The MT1 receptor is known to inhibit second messenger cyclic adenosine monophosphate (cAMP) signaling through receptor-coupling to inhibitory G-proteins (G(i) ). Much less is known about the MT2 receptor, but it has also been implicated in signaling via G(i) -proteins. In rat pancreatic ß-cells, it has recently been reported that the MT2 receptor plays an inhibitory role in the cyclic guanosine monophosphate (cGMP) pathway. This study addresses the signaling features of the constitutively expressed human recombinant MT2 receptor (hMT2) and its impact on insulin secretion, using a rat insulinoma ß-cell line (INS-1). On the basis of a specific radioimmunoassay, insulin secretion was found to be more strongly reduced in the clones expressing hMT2 than in INS-1 controls, when incubated with 1 or 100 nm melatonin. Similarly, cAMP and cGMP levels, measured by specific enzyme-linked immunosorbent assays (ELISAs), were reduced to a greater extent in hMT2 clones after melatonin treatment. In hMT2-expressing cells, the inhibitory effect of melatonin on insulin secretion was blocked by pretreatment with pertussis toxin, demonstrating the coupling of the hMT2 to G(i) -proteins. These results indicate that functional hMT2 expression leads to the inhibition of cyclic nucleotide signaling and a reduction in insulin release. Because genetic variants of the hMT2 receptor are considered to be risk factors in the development of type 2 diabetes, our results are potentially significant in explaining and preventing the pathogenesis of this disease.

Impact of cochlear ablation on calbindin and synaptophysin in the gerbil medial nucleus of the trapezoid body before hearing onset.

Spontaneous bursting activity is already generated in the cochlea before hearing onset and represents an important condition of the functional and anatomical organization of auditory brainstem nuclei. In the present study, cochlea ablation induced changes were characterized in auditory brainstem nuclei indirectly innervated by auditory nerve fibers before hearing onset. In Meriones unguiculatus immunohistochemical labeling of calbindin-D28k (CB) and synaptophysin (SYN) were performed. The influence of cochlea-ablation on CB or SYN was analyzed by considering their differential immunoreaction during development. During the normal postnatal development, CB was first detected in somata of the medial nucleus of the trapezoid body (MNTB) at postnatal day (P)4. The immunoreaction increased gradually in parallel to the appearance of CB-immunoreactive terminal fields in distinct superior olivary complex (SOC) nuclei. Cochlear removal at P5 or P9 in animals with 24 and 48 h survival times resulted in an increase in somatic CB-labeling in the lesioned MNTB including terminal fields compared to the non-lesioned MNTB. SYN-immunolabeling was first detected at P0 and began to strongly encircle the MNTB neurons at P4. A further progression was observed with age. Cochlear ablation resulted in a significant reduction of SYN-labeled MNTB areas of P5-cochlea-ablated gerbils after 48 h post-lesion. In P9 cochlea-ablated gerbils, a redistribution of SYN-positive terminals was seen after 24 and 48 h. Taken together, the destruction of cochlea differentially influences CB- and SYN-labeling in the MNTB, which should be considered in association with different critical periods before hearing onset.

Distribution patterns of calcium-binding proteins in pancreatic tissue of non-diabetic as well as type 2 diabetic rats and in rat insulinoma beta-cells (INS-1).

The present study dealt with the localization of different calcium-binding proteins (CaBPs) in the pancreatic tissue of non-diabetic and diabetic rats and in rat insulinoma beta-cells (INS-1). Transcripts of CaBPs displayed different expression levels in rat pancreatic tissue and INS-1 cells. Immunohistochemistry demonstrated that three of these proteins, calmodulin, calreticulin and calbindin-D28k, were located predominantly in the pancreatic islets (in both alpha- and beta-cells) of rats, showing weaker labeling of exocrine tissue. Secretagogin was exclusively found within islets. All CaBPs were also immunohistochemically detected in INS-1 cells. Immunohistochemical analysis demonstrates differences in CaBP distributions when comparing the pancreatic tissues of diabetic Goto-Kakizaki rats and non-diabetic Wistar rats. Pancreatic tissue in type 2 diabetic Goto-Kakizaki rats showed significantly higher transcript levels of all CaBPs compared to those in Wistar rats. These results indicate that alterations of CaBPs in pancreatic islets are associated with metabolic disturbances related to type 2 diabetes.

Impact of cochlear ablation on calretinin and synaptophysin in the gerbil anteroventral cochlear nucleus before the hearing onset.

Mammalian auditory system undergoes many structural and functional modifications during postnatal development, which are dependent on the relationship between auditory nerve fibers and their nuclei. In the present study, the cochlea of Meriones unguiculatus was ablated unilaterally on postnatal day 5 or 9 (P5 or P9), before the onset of hearing. Histochemical analysis of synaptophysin (SYN) and calretinin (CR) in anterior anteroventral cochlear nucleus (AVCN-A) was performed to analyze whether unilateral cochlea ablation induces changes in the auditory terminal endings and somata of spherical bushy cells (SBCs). During the period of postnatal development, CR-labeling was evident in somata of SBCs and in auditory nerve terminals. SYN was most apparent in puncta encircled cell bodies, progressing with age. Cochlear removal at P5 induced a decrease in CR-labeling in SBCs somata 6 h and 48 h post-lesion; whereas, ablation at P9 increased the somatic CR-labeling in the lesioned AVCN-A after 24 and 48 h post-lesion. The SYN-labeled synaptic puncta were remarkably reduced in the AVCN-A of P5- and P9-cochlea-ablated gerbils with stronger effects in P5 animals (a 50% reduction after 48 h). Interestingly, a significant increase in the SYN-immunolabeled puncta was found after 48 h compared to 24 h in the lesioned AVCN-A of P9 gerbils, indicating reactive synaptogenesis. Our study shows, that following the destruction of the cochlea at different postnatal periods, the CR- and SYN-labeling are differentially influenced in the AVCN-A, which in turn coincides with different critical developmental periods before the onset of hearing.

Differential and day-time dependent expression of nuclear receptors RORα, RORß, RORγ and RXRα in the rodent pancreas and islet.

The retinoic-acid-related receptor family of orphan receptors (RORs) act as transcriptional activators or repressors. One of their functions involves integrated actions within circadian oscillators, particularly of the periphery. The present paper describes differential expression of the orphan receptors RORα, RORß and RORγ and of the nuclear retinoid receptor RXRα in the pancreas and islet of rats. Immunohistochemistry of rodent islets detected nuclear receptor expression. The RORα and RORß signals were visualised in α-cells, whereas that of RORγ was largely confined to ß-cells. RXRα was expressed throughout the islets. Quantitative RT-PCR revealed circadian expression in the rat pancreas for RORγ, RORα and RXRα, but not for RORß. Circadian expression of RORγ mRNA was verified in mouse pancreas and in rat INS-1 ß cells by serum shock experiments. The results point to differential and circadian expression and thus cell-type-specific functions of RORα and RORγ in islet cells secreting glucagon or insulin.

Anatomical changes after pneumonectomy.

Pneumonectomy is associated with many diverse post-operative conditions, e.g. hydropneumothorax, diaphragm elevation, progressive mediastinal displacement, thorax wall deformation, and hydrothorax. By means of imaging procedures, such pneumonectomy-related anatomical changes can easily be determined; here we summarize some of the common diagnostic findings and in addition report the case of a 100-year-old woman, who underwent left pneumonectomy at the age of 47, survived for another 53 years with only one lung and then became body donor to our department. Investigation of the cadaver revealed that, compared to similar-aged individuals still having both lungs, mediastinal structures had been displaced to the side of the missing lung. In addition, the remaining lung had herniated across the midline to a position anterior to the heart. Histological examination of the remaining lung tissue revealed changes comparable to those generally expected in lungs of individuals of the same age-group; tissue changes directly associated with pneumonectomy could not be observed. The findings document anatomical alterations that arise physiologically due to pneumonectomy if no pathological complications occur.