Jes Thorn Clausen

Hypothalamic CART is a new anorectic peptide regulated by leptin

The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

The dynamics of gene expression of interleukin-19 and interleukin-20 and their receptors in psoriasis

Background  Interleukin (IL)‐20 and IL‐19 are recently discovered members of the IL‐10 family of cytokines. The skin of transgenic mice overexpressing IL‐20 shows histological changes resembling some of those seen in psoriasis, i.e. thickened epidermis, hyperkeratosis and a compact stratum corneum. IL‐19 and IL‐20, as well as their receptor complexes, IL‐20Rα/IL‐20Rβ and IL‐22Rα/IL‐20Rβ, are expressed in human skin.

The hypothalamic satiety peptide CART is expressed in anorectic and non-anorectic pancreatic islet tumors and in the normal islet of Langerhans

The hypothalamic satiety peptide CART (cocaine and amphetamine regulated transcript) is expressed at high levels in anorectic rat glucagonomas but not in hypoglycemic insulinomas. However, a non‐anorectic metastasis derived from the glucagonoma retained high CART expression levels and produced circulating CART levels comparable to that of the anorectic tumors. Moreover, distinct glucagonoma lines derived by stable HES‐1 transfection of the insulinoma caused severe anorexia but retained low circulating levels of CART comparable to that of insulinoma bearing or control rats. Islet tumor associated anorexia and circulating CART levels are thus not correlated, and in line with this peripheral administration of CART (5–50 mg/kg) produced no effect on feeding behavior. In the rat two alternatively spliced forms of CART mRNA exist and quantitative PCR revealed expression of both forms in the hypothalamus, in the different islet tumors, and in the islets of Langerhans. Immunocytochemistry as well as in situ hybridization localized CART expression to the somatostatin producing islet D cell. A potential endocrine/paracrine role of islet CART remains to be clarified.

Interleukin-20 plays a critical role in maintenance and development of psoriasis in the human xenograft transplantation model

Background  Interleukin (IL)‐20 is a recently discovered cytokine displaying increased levels in psoriatic lesions. Interestingly, IL‐20 levels decrease with antipsoriatic treatment, correlating with clinical improvement. However, the role of IL‐20 in the aetiology of psoriasis is unknown.

Importance of cocaine- and amphetamine-regulated transcript peptide in the central nucleus of amygdala in anxiogenic responses induced by ethanol withdrawal.

We studied the involvement of cocaine- and amphetamine-regulated transcript peptide (CART) in the central nucleus of amygdala (CeA), lateral bed nucleus of the stria terminalis (BNSTl) and nucleus accumbens shell (AcbSh) in generation of ethanol withdrawal symptoms, with particular focus on anxiety-like behavior using a social interaction test. Administration of CART (54–102) into the lateral ventricle (50 and 100 ng) and bilaterally in the CeA (10 and 20 ng) caused a significant reduction in social interaction, suggesting an anxiogenic action of the peptide. Chronic ethanol treatment for 15 days followed by withdrawal precipitated an anxiogenic response at 24 h that was attenuated by intracerebroventricular (5 μl) and intra-CeA (1 μl) administration of antibodies against CART (1 : 500 dilution). An immunocytochemistry protocol was employed to study the response of the endogenous CART system in the CeA following chronic ethanol withdrawal. At 0 h ethanol withdrawal, CART immunoreactivity was apparent in few fibers and the profile was similar to that in the pair-fed control rats. Twenty-four hours following ethanol withdrawal, a highly significant increase (P<0.001) in CART immunoreactivity was noticed in the CeA, which returned to normal 48 and 72 h post-withdrawal. Similar doses of CART or CART antibody injected bilaterally into the BNSTl or AcbSh produced no response in the social interaction test. Furthermore, the CART immunoreactivity profile did not change at the post-withdrawal time points in each of these brain sites. We suggest that CART may mediate the early signs of anxiety-like behavior induced by ethanol withdrawal within the neuroanatomical framework of the CeA.

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

Immunoassays of human trefoil factors 1 and 2: measured on serum from patients with inflammatory bowel disease

Background: The trefoil factors (TFF1–3) are cysteine‐rich peptides expressed in the gastrointestinal tract where they play a critical role in mucosal protection and repair. The expression is up‐regulated at sites of ulceration in various chronic inflammatory diseases. Recently, we presented an ELISA method for measurement of TFF3. The aims of the present study were to develop and evaluate ELISAs for the other two known human trefoil peptides, TFF1 and TFF2, and to carry out a cross‐sectional study on serum TFF levels in patients with inflammatory bowel disease (IBD). Methods: The TFF1‐ELISA was based on two polyclonal rabbit antibodies and the TFF2‐ELISA on a monoclonal mouse antibody and a polyclonal rabbit antibody. RhTFF1 and 2 were employed to prepare the calibrators. TFF1–3 were assayed in serum from IBD patients (n=41) and controls (n=13). Results: The TFF1‐ (TFF2‐) ELISA had a detection limit of 3 pmol/L (6 pmol/L) and an analytical imprecision (CVA) of 7.0–8.8 for mean concentrations of 24–120 pmol/L (6.1–8.0 for mean concentrations of 17–77 pmol/L). The central reference intervals (n=300) were 140–1400 pmol/L (37–190 pmol/L). There was no variation with age and menstrual cycle. Food intake reduced concentrations of TFF1 by ∼15%, but did not influence concentrations of TFF2. TFF1 and TFF3 were increased in serum from IBD patients. Conclusions: We have developed assays for measuring TFF1 and TFF2. Finding increased TFF concentrations in serum from IBD patients suggests that measurements of trefoil peptides may be of clinical relevance in IBD.

Methylcholanthrene-induced sarcomas in nude mice have short induction times and relatively low levels of surface MHC class I expression.

In order to study the role of the T‐cell‐mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 μg (0.1%), 40 μg (0.5%) and 400 μg (5%)) of 3‐methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice. A shorter tumor induction time and a higher tumor incidence after treatment with low doses of methylcholanthrene were seen in nude mice than in immunocompetent mice, indicating that they have a lower resistance to the carcinogen. Contrary to expectations we found that the MHC class I expression of tumors from nude mice was lower than that of tumors from normal mice. Higher surface expression of MHC class I was demonstrated on high dose tumors from normal mice than on low dose tumors from normal mice. The cellular composition of the individual tumors raised in nude mice was more heterogeneous with respect to MHC class I expression. Since the mice differ genetically only with respect to the nu gene, these results indicate that a lack of T‐cell‐mediated defense mechanisms may confer upon the bearer a lower resistance to 3‐methylcholanthrene and a different MHC profile of the ensuing tumor.

Immunohistochemical localization of cocaine- and amphetamine-regulated transcript peptide in the brain of the catfish, Clarias batrachus (Linn.)

The organization of cocaine‐ and amphetamine‐regulated transcript peptide (CARTp, 54–102) immunoreactivity was investigated in the brain of the catfish, Clarias batrachus. CARTp‐immunoreactivity was observed in several granule cells of the olfactory bulbs, in dot‐like terminals around mitral cells, and in the fibers of the medial olfactory tracts. While several groups of discrete cells in the telencephalon showed CARTp‐immunoreactivity, the immunostained fibers were widely distributed in the area dorsalis and ventralis telencephali. Immunoreactivity was seen in several periventricular and a few magnocellular neurons, and in a dense fiber network throughout the preoptic area. Varying degrees of immunoreactive fibers were seen in the periventricular region in the thalamus, hypothalamus, and pituitary. Some neurons in the nucleus preglomerulosus medialis and lateralis, central nucleus of the inferior lobes, nucleus lobobulbaris of the posterior tuberculum, and nucleus recessus posterioris showed distinct CARTp‐immunoreactivity. Considerable immunoreactivity was seen in the optic tectum, rostral torus semicircularis, central pretectal area, and granule cells of the cerebellum. While only isolated immunoreactive cells were seen at three distinct sites in the metencephalon, a fiber network was seen in the facial and vagal lobes and periventricular and ventral regions of the medulla oblongata. The pattern of the CARTp distribution in the brain of C. batrachus suggests that it may play an important role in the processing of sensory information, the regulation of hormone secretion by hypophysial cell types, and motor and vegetative function. Finally, as in other animal species, CARTp seems to play a role in the processing of gustatory information. J. Comp. Neurol. 502:215–235, 2007.

Identification of a novel integral plasma membrane protein induced during adipocyte differentiation.

Adipocyte differentiation is co-ordinately regulated by several transcription factors and is accompanied by changes in the expression of a variety of genes. Using mRNA differential display analysis, we have isolated a novel mRNA, DD16, specifically induced during the course of adipocyte differentiation. DD16 mRNAs are present in several tissues, but among the tissues tested, a remarkably higher level of expression was found in white adipose tissue. The DD16 cDNA encoded a polypeptide of 415 amino acids containing a single N-glycosylation site and an N-terminal hydrophobic stretch of 19 amino acids forming a transmembrane segment, indicating that DD16 is a glycosylated membrane-bound protein. Polyclonal antibodies raised against the DD16 peptide detected immunoreactive DD16 in membrane fractions, notably the plasma membrane. Association of DD16 with the plasma membrane was further confirmed by biotinylation studies of cell surface proteins, suggesting that DD16 is an integral plasma membrane protein. Therefore we propose to give DD16 the name APMAP (Adipocyte Plasma Membrane-Associated Protein). Although the biological function of this polypeptide is presently unknown, our data suggest that APMAP may function as a novel protein involved in the cross-talk of mature adipocytes with the environment.