No abstract

The effects of vascular factors on the nervous system are still poorly investigated. Angiopoietin-1 (Ang-1), an endothelial cell growth factor with influences on blood vessel stabilization, has been recently reported to prevent apoptosis in a neuroblastoma cell line via a pathway dependent on Tie-2 receptor. The present study focuses on the effect of Ang-1 on cultured dorsal root ganglion (DRG) cells isolated from 1-day-old rats. Three-day-old DRG cultures were exposed to Ang-1 treatment under serum-free condition for another 5 days and stained with antibodies against neurofilament (NF) 200 protein. Neurite length and density increased compared with those of controls. Double-immunofluorescence staining demonstrated the co-localization of the Tie-2 receptor in some NF-200-positive perikarya. The reverse transcription/polymerase chain reaction technique identified Tie-2 receptor mRNA in intact DRG and in Ang-1-stimulated DRG cell cultures, but not in a Schwann cell line or in primary astrocyte cultures. Western blotting confirmed that the expression of NF 68 protein in cultures treated with Ang-1 or nerve growth factor was higher than that in cultures treated with medium alone. When the Tie-2 receptor was blocked with anti-Tie-2 receptor antibody, neurite outgrowth was severely impeded. Induction of trkA-receptor protein expression was observed to be dependent on the presence of Tie-2 receptors. We conclude that Ang-1 promotes neurite outgrowth from DRG cells positive for Tie-2 receptor. The signalling pathway appears to involve transactivation of the trkA receptor.

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin-1 (Ang-1). We further considered initial hints that Ang-1-induced neuritogenesis involves transactivation of the high-affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3-L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang-1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang-1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high-affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang-1 and NGF in the control of neuritogenesis. DRG-adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD-95 correlated with Ang-1 levels in culture. It is concluded that adipocyte-secreted Ang-1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang-1 appears to play a physiological role in WAT plasticity.

The morphology of sciatic nerves from leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice, both models for obesity, peripheral diabetic neuropathy, and the metabolic syndrome, has yet to be examined for changes in nerve fibers and in endoneural microvessels. Sciatic nerves from three groups of 4-month-old mice (WT C57BL6, ob/ob, and db/db) were investigated. In ultrathin sections, the thickness of myelin sheaths was significantly reduced in small, medium-sized, and large axons of db/db mice compared with WT mice. In ob/ob mice, only large fibers showed a decrease in myelin sheath thickness. The number of nonmyelinated nerve fibers was lower in ob/ob mice than in the db/db group. A thickened basal lamina of Schwann cells occurred in the ob/ob group only. In contrast, the basement membrane of endoneural microvessels was thickened in both obese groups. For this reason, laminin expression in Western blot analysis was lower in the db/db group than in the ob/ob one. Endoneural microvessels, which had been injected with fluorescein isothiocyanate, depicted signs of vasodilatation in the ob/ob and vasoconstriction in db/db mice. Endoneural vessels displayed two receptors of oxLDL. LOX-1 was strongly expressed in db/db mice, whereas TLR4 was at its maximum in the ob/ob group. We conclude that changes in nerve fibers and in endoneural microvessels are present in sciatic nerve of both mouse models of type 2 diabetes. Upregulation of oxLDL-dependent receptors in endoneural microvessels might be connected to different degrees of oxidative stress in severe diabetic db/db mice and in the mild diabetic ob/ob group.

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.