Serotonin improves glucose metabolism by Serotonylation of the small GTPase Rab4 in L6 skeletal muscle cells.

BACKGROUND: Serotonin (5-HT) improves insulin sensitivity and glucose metabolism, however, the underlying molecular mechanism has remained elusive. Previous studies suggest that 5-HT can activate intracellular small GTPases directly by covalent binding, a process termed serotonylation. Activated small GTPases have been associated with increased GLUT4 translocation to the cell membrane. Therefore, we investigated whether serotonylation of small GTPases may be involved in improving Insulin sensitivity and glucose metabolism. METHODS: Using fully differentiated L6 rat skeletal muscle cells, we studied the effect of 5-HT in the absence or presence of insulin on glycogen synthesis, glucose uptake and GLUT4 translocation. To prove our L6 model we additionally performed preliminary experiments in C2C12 murine skeletal muscle cells. RESULTS: Incubation with 5-HT led to an increase in deoxyglucose uptake in a concentration-dependent fashion. Accordingly, GLUT4 translocation to the cell membrane and glycogen content were increased. These effects of 5-HT on Glucose metabolism could be augmented by co-incubation with insulin and blunted by co incubation of 5-HT with monodansylcadaverine, an inhibitor of protein serotonylation. In accordance with this observation, incubation with 5-HT resulted in serotonylation of a protein with a molecular weight of approximately 25 kDa. We identified this protein as the small GTPase Rab4, the activity of which has been shown to be stimulated by both insulin signalling and serotonylation. CONCLUSION: Our data suggest that 5-HT elicits its beneficial effects on Glucose metabolism through serotonylation of Rab4, which likely represents the converging point between the insulin and the 5-HT signalling cascades.

Postprandial lipemia induces pancreatic α cell dysfunction characteristic of type 2 diabetes: studies in healthy subjects, mouse pancreatic islets, and cultured pancreatic α cells.

BACKGROUND: Type 2 diabetes is associated with pancreatic α cell dysfunction, characterized by elevated fasting plasma glucagon concentrations and inadequate postprandial glucose- and insulin-induced suppression of glucagon secretion. The cause and the underlying mechanisms of α cell dysfunction are unknown. OBJECTIVE: Because Western dietary habits cause postprandial lipemia for a major part of a day and, moreover, increase the risk of developing type 2 diabetes, we tested the hypothesis that postprandial lipemia with its characteristic elevation of triglyceride-rich lipoproteins (TGRLs) might cause pancreatic α cell dysfunction. DESIGN: In a crossover study with 7 healthy volunteers, 2 experiments using 2 fat-enriched meals were performed on each volunteer; meal 1 was designed to increase plasma concentrations of both TGRLs and nonesterified fatty acids and meal 2 to increase TGRLs only. Intravenous glucose boli were injected at 0800 after an overnight fast and postprandially at 1300, 3 h after ingestion of a fat-enriched meal. Glucagon concentrations were measured throughout the days of the experiments. In addition to the study in humans, in vitro experiments were performed with mouse pancreatic islets and cultured pancreatic alpha TC 1 clone 9 (αTC1c9) cells, which were incubated with highly purified TGRLs. RESULTS: In humans, postprandial lipemia increased plasma glucagon concentrations and led to an inadequate glucose- and insulin-induced suppression of glucagon. There was no difference between the 2 meal types. In mouse pancreatic islets and cultured pancreatic αTC1c9 cells, purified postprandial TGRLs induced abnormalities in glucagon kinetics comparable with those observed in humans. The TGRL-induced α cell dysfunction was due to reduced γ-aminobutyric acid A receptor activation in pancreatic α cells. CONCLUSION: We concluded that postprandial lipemia induces pancreatic α cell dysfunction characteristic of type 2 diabetes and, therefore, propose that pancreatic α cell dysfunction could be viewed, at least partly, as a postprandial phenomenon.

Quantitative analysis of volatile organic compounds released and consumed by rat L6 skeletal muscle cells in vitro.

Knowledge of the release of volatile organic compounds (VOCs) by cells provides important information on the origin of VOCs in exhaled breath. Muscle cells are particularly important, since their release of volatiles during the exertion of an effort contributes considerably to breath concentration profiles. Presently, the cultivation of human skeletal muscle cells is encountering a number of obstacles, necessitating the use of animal muscle cells in in vitro studies. Rat L6 skeletal muscle cells are therefore commonly used as a model for studying the molecular mechanisms of human skeletal muscle differentiation and functions, and facilitate the study of the origin and metabolic fate of the endogenously produced compounds observed in breath and skin emanations. Within this study the production and uptake of VOCs by rat L6 skeletal muscle cells were investigated using gas chromatography with mass spectrometric detection, combined with head-space needle trap extraction as the pre-concentration technique (HS-NTE-GC-MS). Seven compounds were found to be produced, whereas sixteen species were consumed (Wilcoxon signed-rank test, p < 0.05) by the cells being studied. The set of released volatiles included two ketones (2-pentanone and 2-nonanone), two volatile sulphur compounds (dimethyl sulfide and methyl 5-methyl-2-furyl sulphide), and three hydrocarbons (2-methyl 1-propene, n-pentane and isoprene). Of the metabolized species there were thirteen aldehydes (2-propenal, 2-methyl 2-propenal, 2-methyl propanal, 2-butenal, 2-methyl butanal, 3-methyl butanal, n-pentanal, 2-methyl 2-butenal, n-hexanal, benzaldehyde, n-octanal, n-nonanal and n-decanal), two esters (n-propyl propionate and n-butyl acetate), and one volatile sulphur compound (dimethyl disulfide). The possible metabolic pathways leading to the uptake and release of these compounds by L6 cells are proposed and discussed. An analysis of the VOCs showed them to have huge potential for the identification and monitoring of some molecular mechanism and conditions.

Postprandial triglyceride-rich lipoproteins induce hepatic insulin resistance in HepG2 cells independently of their receptor-mediated cellular uptake.

Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance with the molecular basis of this association being not well understood. Here we studied the effect of hepatic triglyceride accumulation induced by postprandial triglyceride-rich lipoproteins (TGRL) on hepatic insulin sensitivity in HepG2 cells. Incubation of HepG2 cells with purified TGRL particles induced hepatocellular triglyceride accumulation paralleled by diminished insulin-stimulated glycogen content and glycogen synthase activity. Accordingly, insulin-induced inhibition of glycogen synthase phosphorylation as well as insulin-induced GSK-3 and AKT phosphorylation were reduced by TGRL. The effects of TGRL were dependent on the presence of apolipoproteins and more pronounced for denser TGRL. Moreover, TGRL effects required the presence of heparan sulfate-proteoglycans on the cell membrane and lipase activity but were independent of the cellular uptake of TGRL particles by receptors of the LDL receptor family. We suggest postprandial lipemia to be an important factor in the pathogenesis of NAFLD.

Association of the canine guidance angle with maxillary and mandibular intercanine widths and anterior alignment relapse: Extraction vs nonextraction treatment.

INTRODUCTION: Long-term stability is an important measure of the success of orthodontic treatment. Research in the 1970s suggested that premolar extraction treatment had poor stability over the long term. The purpose of this prospective follow-up study was to investigate changes in intercanine widths and the irregularity index during the postretention phase in patients treated with and without extractions. Associations between the maxillary canine guidance angle and the mandibular intercanine widths and the mandibular irregularity index were also examined. METHODS: Two groups of 30 patients each, with and without premolar extractions, were studied and the results compared. Records were taken at pretreatment, at bracket removal, at the end of retention, and out of retention. The times out of retention were 6.3 years for the nonextraction group and 6.5 years for the extraction group. Stone casts were mounted on an articulator with an anatomical facebow and a central wax record. The measurements were made with a 3-dimensional digitizer. RESULTS: Maxillary and mandibular intercanine distances behaved differently. The mandibular intercanine distance showed a net decrease between pretreatment and follow-up, whereas the maxillary arch had a net increase. The maxillary canine guidance angle at the end of retention, measured to the axis-orbital plane, was highly associated with relapse of mandibular anterior alignment and change of the mandibular intercanine distance. CONCLUSIONS: Methods are needed that consider the anatomical configuration of the teeth for bracket selection before treatment. Individualized brackets (torque) should then be coordinated with the guidance angle of the maxillary teeth.

Hypoxia up-regulates the angiogenic cytokine secretoneurin via an HIF-1alpha- and basic FGF-dependent pathway in muscle cells.

Expression of angiogenic cytokines like vascular endothelial growth factor is enhanced by hypoxia. We tested the hypothesis that decreased oxygen levels up-regulate the angiogenic factor secretoneurin. In vivo, muscle cells of mouse ischemic hind limbs showed increased secretoneurin expression, and inhibition of secretoneurin by a neutralizing antibody impaired the angiogenic response in this ischemia model. In a mouse soft tissue model of hypoxia, secretoneurin was increased in subcutaneous muscle fibers. In vitro, secretoneurin mRNA and protein were up-regulated in L6 myoblast cells after exposure to low oxygen levels. The hypoxia-dependent regulation of secretoneurin was tissue specific and was not observed in endothelial cells, vascular smooth muscle cells, or AtT20 pituitary tumor cells. The hypoxia-dependent induction of secretoneurin in L6 myoblasts is regulated by hypoxia-inducible factor-1alpha, since inhibition of this factor using si-RNA inhibited up-regulation of secretoneurin. Induction of secretoneurin by hypoxia was dependent on basic fibroblast growth factor in vivo and in vitro, and inhibition of this regulation by heparinase suggests an involvement of low-affinity basic fibroblast growth factor binding sites. In summary, our data show that the angiogenic cytokine secretoneurin is up-regulated by hypoxia in muscle cells by hypoxia-inducible factor-1alpha- and basic fibroblast growth factor-dependent mechanisms.

Resistin impairs basal and insulin-induced glycogen synthesis by different mechanisms.

In the present study, we investigated the mechanisms by which resistin (100 nM, 1 h) affects glycogen synthesis in L6 skeletal muscle cells. The activity of glycogen synthase, the major enzyme in glycogen synthesis, is determined by both its covalent phosphorylation and allostery through intracellular glucose-6-phosphate. Covalent phosphorylation of glycogen synthase was not altered by resistin and, accordingly, phosphorylation of GSK-3alpha/beta and Akt remained unchanged. The rate of glucose-6-phosphate formation, however, was decreased by resistin both in the absence and presence of insulin; in the absence of insulin, resistin decreased glucose-6-phosphate formation by reducing hexokinase type I activity without affecting glucose uptake; by contrast, in the presence of insulin, resistin decreased glucose-6-phosphate formation by reducing the Vmax of glucose uptake without changing hexokinase type I activity. In conclusion, short-term resistin incubation impairs glycogen synthesis by reducing the rate of glucose-6-phosphate formation involving, however, differential mechanisms in basal and insulin-stimulated states.

A gel filtration assay to determine glycogen synthase activity.

We developed a gel filtration assay for the determination of glycogen synthase activity in cultured cells or tissue homogenates. Compared to the commonly used filter paper assay, the gel filtration assay resulted in a more than 5-fold reduction of background levels leading to an--at least--twofold increase in precision. These benefits allow the gel filtration method to detect differences of +/-5% in enzyme activity out of 300 microg total cell protein. In addition to high precision and sensitivity, the method's additional salient advantages include lesser expenditure of time and labour and reduced exposure time of the personnel to radioactivity.

Axiographic findings in patients undergoing orthodontic treatment with and without premolar extractions.

Mechanical axiography was performed on 49 (37 female, 12 male) patients prior to orthodontic treatment, after removal of the fixed appliance, and at the end of retention. Twenty-five subjects (mean age 12.8 years) underwent orthodontic treatment without premolar extractions (group 1) and 24 subjects (mean age 13.5 years) with premolar extractions (group 2). The axiographic tracings of the protrusive movements were analysed using a digitizer and specially designed software. A statistically significant increase (P < 0.05) in the values for horizontal condylar inclination (HCI) was found for both groups over the entire observation period. Group 1 showed a higher increase (P < 0.05) between the beginning of treatment and removal of the brackets, and group 2 between bracket removal and the end of retention. At the end of retention, a similar increase in HCI values was found for both groups. Over the observation period, the frequency of pathological axiographic findings decreased, which seems to be a positive effect of orthodontic treatment.

Secretoneurin, an angiogenic neuropeptide, induces postnatal vasculogenesis.

BACKGROUND: Induction of postnatal vasculogenesis, the mobilization of bone marrow-derived endothelial progenitor cells and incorporation of these cells into sites of blood vessel formation, is a well-known feature of angiogenic cytokines such as vascular endothelial growth factor. We hypothesized that the angiogenic neuropeptide secretoneurin induces this kind of neovascularization. METHODS AND RESULTS: Secretoneurin induced mobilization of endothelial progenitor cells to sites of vasculogenesis in vivo in the cornea neovascularization assay. Progenitor cells were incorporated into vascular structures or were located adjacent to them. Systemic injection of secretoneurin led to increase of circulating stem cells and endothelial progenitor cells. In vitro secretoneurin induced migration, exerted antiapoptotic effects, and increased the number of these cells. Furthermore, secretoneurin stimulated the mitogen-activated protein kinase system, as shown by phosphorylation of extracellular signal-regulated kinase, and activated the protein kinase B/Akt pathway. Activation of mitogen-activated protein kinase was necessary for increase of cell number and migration, whereas Akt seemed to play a role in migration of endothelial progenitor cells. CONCLUSIONS: These data show that the angiogenic neuropeptide secretoneurin stimulates postnatal vasculogenesis by mobilization, migration, and incorporation of endothelial progenitor cells.

Three-dimensional dental arch and palatal form changes after extraction and nonextraction treatment. Part 1. Arch length and area.

The purpose of this study was to investigate changes in arch length, arch area, and irregularity index in patients treated with and without premolar extractions. Records collected at pretreatment, at bracket removal, at the end of retention, and 5 years out of retention were examined. Stone casts were mounted on an articulator with an anatomic face-bow and a central wax record, and measurements were made with a 3-dimensional digitizer. In general, the maxillary arch exhibited less relapse tendency than did the mandibular arch for both patient groups. In general, the extraction group showed the same relapse tendency as the nonextraction group.

Three-dimensional dental arch and palatal form changes after extraction and nonextraction treatment. Part 2. Palatal volume and height.

The purpose of this study was to investigate the changes in palatal volume and palatal height in patients treated with and without premolar extractions. Records were collected at pretreatment, at bracket removal, at the end of retention, and 5 years out of retention. Stone casts were mounted in a SAM 2 articulator with an anatomic face-bow and a central wax record, and measurements were made with a 3-dimensional digitizer. The hypothesis, that orthodontic treatment with premolar extractions changes the palatal form, was verified. Increases in palatal volume and height were demonstrated in the nonextraction group. The extraction group showed a decrease in palatal volume but could compensate for some loss by an increase in volume in the anterior segment.

Three-dimensional dental arch and palatal form changes after extraction and nonextraction treatment. Part 3. Transversal and sagittal palatal form.

The purpose of this study was to investigate changes in the palatal form in patients treated with and without premolar extractions. Records collected at pretreatment, at bracket removal, at the end of retention, and 5 years out of retention were examined. Stone casts were mounted on an articulator with an anatomic face-bow and a central wax record, and measurements were made with a 3-dimensional digitizer. The hypothesis that orthodontic treatment with premolar extractions changes the palatal form was verified.