Differential adipose tissue inflammatory state in obese nondiabetic Zucker fatty rats compared to obese diabetic zucker diabetic fatty rats.

Adipose tissue (AT) inflammation is linked to the pathogenesis of diabetes in obesity. Here, we compare the AT inflammatory state of 2 animal models of obesity and obesity plus diabetes, respectively. Obese nondiabetic ZF rats exhibited a trend towards increased proportions of CD11b positive cells in the adipose tissue stroma vascular fraction suggesting a state of increased AT inflammation compared to their lean littermates, but no alterations in systemic inflammatory parameters. In contrast, obese diabetic ZDF rats exhibited systemic as well as local AT inflammation with elevated levels of circulating Regulated upon Activation, Normal T-cell Expressed and Secreted Protein (Rantes), interleukin 1ß (IL-1ß) and monocyte chemotactic protein 1 (MCP-1), and an increased infiltration of adipose tissue CD11b positive cells. Our data provide a novel phenotypic characterisation of 2 common metabolic animal models and suggest an association of obesity with local inflammation in adipose tissue, and an association of diabetes with local inflammation in adipose tissue plus systemic inflammation. AT inflammation in obesity might therefore initiate a process that above a certain limits finally results in systemic inflammation and diabetes.

Gene expression profiling in skeletal muscle of Zucker diabetic fatty rats: implications for a role of stearoyl-CoA desaturase 1 in insulin resistance.

AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes. Therefore, we sought to identify and validate genes involved in the development of insulin resistance in skeletal muscle. MATERIALS: Differentially regulated genes in skeletal muscle of male obese insulin-resistant, and lean insulin-sensitive Zucker diabetic fatty (ZDF) rats were determined using Affymetrix microarrays. Based on these data, various aspects of glucose disposal, insulin signalling and fatty acid composition were analysed in a muscle cell line overexpressing stearoyl-CoA desaturase 1 (SCD1). RESULTS: Gene expression profiling in insulin-resistant skeletal muscle revealed the most pronounced changes in gene expression for genes involved in lipid metabolism. Among these, Scd1 showed increased expression in insulin-resistant animals, correlating with increased amounts of palmitoleoyl-CoA. This was further investigated in a muscle cell line that overexpressed SCD1 and accumulated lipids, revealing impairments of glucose uptake and of different steps of the insulin signalling cascade. We also observed differential effects of high-glucose and fatty acid treatment on glucose uptake and long-chain fatty acyl-CoA profiles, and in particular an accumulation of palmitoleoyl-CoA in cells overexpressing SCD1. CONCLUSIONS/INTERPRETATION: Insulin-resistant skeletal muscle of ZDF rats is characterised by a specific gene expression profile with increased levels of Scd1. An insulin-resistant phenotype similar to that obtained by treatment with palmitate and high glucose can be induced in vitro by overexpression of SCD1 in muscle cells. This supports the hypothesis that elevated SCD1 expression is a possible cause of insulin resistance and type 2 diabetes.

Functional cooperation of Epstein-Barr virus nuclear antigen 2 and the survival motor neuron protein in transactivation of the viral LMP1 promoter.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for viral transformation of B cells and transactivates cellular and viral target genes by binding RBPJkappa tethered to cognate promoter elements. EBNA2 interacts with the DEAD-box protein DP103 (DDX20/Gemin3), which in turn is complexed to the survival motor neuron (SMN) protein. SMN is implicated in RNA processing, but a role in transcriptional regulation has also been suggested. Here, we show that DP103 and SMN are complexed in B cells and that SMN coactivates the viral LMP promoter in the presence of EBNA2 in reporter gene assays and in vivo. Subcellular localization studies revealed that nuclear gems and/or coiled bodies containing DP103 and SMN are targeted by EBNA2. Protein-protein interaction experiments demonstrated that DP103 binds to SMN exon 6 and that both EBNA2 and SMN interact with the C terminus of DP103. Furthermore, a DP103 binding-deficient SMN mutant was released from nuclear gems and/or coiled bodies and further enhanced coactivation. In addition, impaired transactivation of a DP103 binding-deficient EBNA2 mutant was rescued by overexpression of SMN. Testing different promoter constructs in luciferase assays showed that RBPJkappa is required but not sufficient for coactivation by EBNA2 and SMN. Overall, our data suggest that EBNA2 might target spliceosomal complexes by binding to DP103, thereby releasing SMN which subsequently exerts a coactivational function within the RNA-polymerase II transcription complex on the LMP1 promoter.

A potential NES of the Epstein-Barr virus nuclear antigen 1 (EBNA1) does not confer shuttling.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) is a multifunctional protein involved in the replication and maintenance of the viral episome. We identified a potential Rev-like nuclear export signal (NES) which, however, does not confer the export of EBNA1. In the yeast two-hybrid system EBNA1 does not bind to the nuclear exporter Crm1p. In spite of the RNA-binding ability of EBNA1 and its structural homologies to RNA binding proteins like hnRNP U and/or A1, EBNA1 does not shuttle to the cytoplasm in heterokaryon analysis. We propose the function of the RNA binding of EBNA1 in retaining RNAs to the nucleus.

Serotonin immunoreactive boutons form close appositions with respiratory neurons of the dorsal respiratory group in the cat.

The aim of this study was to examine the location of serotonin immunoreactive boutons on both the soma and dendrites of neurons in the dorsal respiratory group by using a combination of intracellular recording and labelling and immunohistochemistry. Inspiratory neurons in the ventrolateral nucleus of the solitary tract (vl-NTS) were intracellularly labelled with horseradish peroxidase (HRP) in anaesthetised adult cats. The morphology of 23 neurons, all antidromically activated from the contralateral C3 spinal segment, was examined. Six neurons displayed pronounced dendritic arborizations outside the vl-NTS, with prominent dorsal and/or medial projections. The dendrites of the remaining neurons were almost entirely confined to the vl-NTS. Intramedullary axon collaterals were detected in four of nineteen examined axons. Serotoninergic fibres were immunohistochemically demonstrated in the NTS, and the apposition of immunoreactive boutons to the HRP-filled neurons examined at the light microscopic level. Boutons were identified in close apposition with the somata, proximal and distal dendrites of these neurons. However, the density of contacts was found to be substantially less than in a previous study of phrenic motoneurons (Lipski et al: Soc. Neurosci. Abst. 14:379, '88; Pilowsky et al: J. Neurosci. in press, '90). The relative paucity of contacts of serotonin immunoreactive boutons with premotor inspiratory neurons of the dorsal respiratory group indicates that the serotoninergic system affects respiratory pathways mainly at the level of respiratory motoneurons or at brainstem sites outside the vl-NTS.

Serotonin immunoreactive boutons make synapses with feline phrenic motoneurons.

In anesthetized cats, phrenic motoneurons were intracellularly labeled with HRP. Immunohistochemistry was used to localize serotonin-like immunoreactivity that was present in numerous boutons and nerve fibers within the ventral horn of the C5 spinal segment. Immunoreactive boutons were frequently found in apposition to phrenic motoneurons, but these close contacts were more common on the dendrites than the cell body. At the electron microscope level, serotonin-immunoreactive boutons were found to make synapses with well-defined postsynaptic densities on proximal and distal dendrites of phrenic motoneurons. These results suggest that serotonin-containing neurons may directly affect the excitability of phrenic motoneurons, mainly through an input onto their extensive dendritic trees.

Synaptic inhibition of phrenic motoneurones evoked by stimulation of the superior laryngeal nerve.

Synaptic responses evoked in phrenic motoneurones (PMNs) by stimulation of the superior laryngeal nerve (SLN) were analysed in anaesthetised cats. Stimulation of the SLN was followed by inhibition of ipsilateral phrenic nerve discharge with the latency of 9.5 +/- 2.3 ms (mean +/- S.D.) and hyperpolarizations of ipsilateral PMN membrane potentials (latency, 8.4 +/- 2.1 ms) which were observed after stimuli applied both during inspiration and expiration. During the injection of Cl ions, the hyperpolarizations were either reversed or flattened in all 28 tested PMNs, thus indicating a direct inhibition. The possibility that the inhibitory postsynaptic potentials are produced by segmental respiratory interneurones is discussed.