Leptin promotes a proinflammatory lipid profile and induces inflammatory pathways in human SZ95 sebocytes.

BACKGROUND: Leptin, the adipocyte-secreted hormone that regulates weight, is known to link lipid metabolism with inflammation in various cell types. However, its role in human sebocytes has not yet been investigated. OBJECTIVES: The purpose of this study was to investigate the effects of leptin in human sebaceous gland biology. METHODS: Expression of the long form of the leptin receptor (Ob-Rb) was detected by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunochemistry. Lipid analysis was by high-performance thin-layer chromatography, gas chromatography-mass spectrometry and time-of-flight mass spectrometer mass detection. Lipid bodies were visualized by BODIPY staining using fluorescent microscopy and measured by flow cytometry. Interleukin (IL)-6 and IL-8 mRNA levels were assessed by real-time qRT-PCR and their release was evaluated by enzyme-linked immunosorbent assay. Cyclooxygenase (COX)-2 and 5-lipooxygenase (LOX) protein expression and phosphorylation of p65 and signal transducer and activator of transcription (STAT)-3 were determined by Western blot analysis. RESULTS: Expression of Ob-Rb was detected in human sebaceous glands and in cultured human SZ95 sebocytes. The treatment of SZ95 sebocytes with leptin led to enlarged intracellular lipid bodies, increased ratios of unsaturated/saturated fatty acids and decreased vitamin E levels. Further supporting a proinflammatory role, leptin induced COX-2 and 5-LOX expression in SZ95 sebocytes and augmented the production of IL-6 and IL-8 cytokines. On leptin treatment, the STAT-3 and nuclear factor-κB pathways were activated, indicating that these known leptin signalling pathways are active in human sebocytes. CONCLUSIONS: Our findings suggest that leptin signalling may be involved in the proinflammatory regulation of sebaceous lipid metabolism and the induction of inflammatory enzymes and cytokines.

Transmitter content, origins and connections of axons in the spinal cord that possess the serotonin (5-hydroxytryptamine) 3 receptor.

Recent evidence suggests that serotonin has pronociceptive actions in the spinal cord when it acts through 5-hydroxytryptamine (5-HT)(3) receptors. Cells and axon terminals which are concentrated in the superficial dorsal horn possess this receptor. We performed a series of immunocytochemical studies with an antibody raised against the 5-HT(3A) subunit in order to address the following questions: 1) Are axons that possess 5-HT(3) receptors excitatory? 2) Are 5-HT(3) receptors present on terminals of myelinated primary afferents? 3) What is the chemical nature of dorsal horn cells that possess 5-HT(3) receptors? 4) Do axons that possess 5-HT(3) receptors target lamina I projection cells? Approximately 45% of 5-HT(3A) immunoreactive boutons were immunoreactive for the vesicular glutamate transporter 2 and almost 80% formed synapse-like associations with GluR2 subunits of the AMPA receptor therefore it is principally glutamatergic axons that possess the receptor. Immunoreactivity was not present on myelinated primary afferent axons labeled with the B-subunit of cholera toxin or those containing the vesicular glutamate transporter 1. Calbindin (which is associated with excitatory interneurons) was found in 44% of 5-HT(3A) immunoreactive cells but other markers for inhibitory and excitatory cells were not present. Lamina I projection cells that possessed the neurokinin-1 receptor were associated with 5-HT(3A) axons but the density of contacts on individual neurons varied considerably. The results suggest that 5-HT(3) receptors are present principally on terminals of excitatory axons, and at least some of these originate from dorsal horn interneurons. The relationship between lamina I projection cells and axons possessing the 5-HT(3) receptor indicates that this receptor has an important role in regulation of ascending nociceptive information.

The neuropeptide FMRFamide can protect cells against apoptosis in the snail digestive gland.

FMRFamide-related peptides are widespread neurotransmitters or neurohormones regulating somatic or visceral motor activity. Some recent data indicate that these neuropeptides may be involved in the control of cell proliferation and apoptosis. In this work we investigated the possible effect of FMRFamide on cell viability in an invertebrate-type proliferating tissue. As a model, we used the midintestinal gland of the snail, Helix lucorum Linnaeus. Immunohistochemistry demonstrated the direct innervation of the gland cells by FMRFamide-containing nerve fibers. Midintestinal glands of snails were injected with 50 microM FMRFamide and the control with sterile deionised water or bovine serum albumin (BSA). Injections were administrated 4 times. Transmission electron microscopy, annexin V-labeling, thiazolyl blue (MTT) viability tests and ploidy analyses were carried out to define the viable/dead cell ratio in the tissue samples. FMRFamide increased the MTT-reduction of tissues, reduced the amount of apoptotic nuclei and annexin V-labeled cells. Deionised water or BSA injection induced cell death. Cell cycle analysis revealed that FMRFamide significantly elevated the amount of cells in G0/G1 phase, but did not induce mitosis. We conclude, that the FMRFamide can be a life-signal for cells, protect them from apoptosis without altering mitosis.

P boutons in lamina IX of the rodent spinal cord express high levels of glutamic acid decarboxylase-65 and originate from cells in deep medial dorsal horn.

Presynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population of GABAergic axon terminals known as P boutons. Despite extensive investigation, the cells that mediate this control have not yet been identified. In this work, we use immunocytochemistry with confocal microscopy and EM to demonstrate that P boutons can be distinguished from other GABAergic terminals in the ventral horn of rat and mouse spinal cord by their high level of the glutamic acid decarboxylase (GAD) 65 isoform of GAD. By carrying out retrograde labeling from lamina IX in mice that express green fluorescent protein under the control of the GAD65 promoter, we provide evidence that the cells of origin of the P boutons are located in the medial part of laminae V and VI. Our results suggest that P boutons represent the major output of these cells within the ventral horn and are consistent with the view that presynaptic inhibition of proprioceptive afferents is mediated by specific populations of interneurons. They also provide a means of identifying P boutons that will be important in studies of the organization of presynaptic control of Ia afferents.

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn.

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.