D2 dopamine receptor (DRD2) gene Taq1A polymorphism and the eating-related psychological traits in eating disorders (anorexia nervosa and bulimia) and obesity.

OBJECTIVE: Food is considered a reinforcing agent, like a variety of substances such as alcohol and other drugs of abuse that produce pleasure. Psychopathological traits related to food intake are demonstrated in eating disorders as in obesity with different genetic aspects for these diseases. Recently, the prevalence of TaqA1 allele has been associated to alcohol, drug abuse and carbohydrate preference. For this reason, the aim of this study was to evaluate if the presence of A1 allele, in eating disorders and obesity, is associated with some particular psycho-pathological characteristics. METHODS: We studied the presence of TaqA1 in Italian subjects affected by obesity (n=71), anorexia (n=28), bulimia (n=20) and in control group (n=54). The Eating Disorders Inventory (EDI test) was used to evaluate the psychological profiles. Patients without alcohol and drugs abuse were selected (>125 ml/day). RESULTS: The A1+ allele, both in A1/A1 and A1/A2 genotypes, was not differently distributed among disease groups; on the contrary two EDI subscales (Drive for thinness and Ineffectiveness) resulted associated with A1+ allele without effect of the eating disease or obesity. CONCLUSION: These results confirm that the presence of A1+ allele is not simply related to body weight but the A1+ allele might be a marker of a genetic psychological condition in people with high risk to develop pathological eating behaviour.

Protective effects of noradrenaline against tumor necrosis factor-alpha-induced apoptosis in cultured rat brown adipocytes: role of nitric oxide-induced heat shock protein 70 expression.

OBJECTIVE: To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-alpha-induced apoptosis of brown adipocytes. DESIGN: Brown fat precursor cells were isolated from young rats; 2.5 million cells were added to each 24-well culture plate and cultured in a defined culture medium. On day 8, the cultured cells were exposed to murine recombinant TNF-alpha and/or cycloheximide (CHX; 10 microg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and/or 10 microM heat shock protein 70 (HSP70) antisense or sense oligomers. MEASUREMENTS: Analysis of DNA fragmentation on agarose gel as a marker of apoptosis; reverse transcriptase-polymerase chain reaction analysis of mRNA levels; Western blotting analysis of protein levels. RESULTS: Pretreatment of primary cultures of rat brown fat cells with micromolar concentrations of NA or the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of HSP70 mRNA and protein, which was associated with cytoprotection against TNF-alpha plus CHX-induced apoptosis. The L-NAME inhibitor of NO synthase activity inhibited both NA-stimulated HSP70 expression and cytoprotection. Furthermore, pretreatment of brown adipocytes with an antisense oligonucleotide to HSP70 antagonized both SNAP- and NA-induced cytoprotection. CONCLUSION: These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-alpha-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.

The putative OP(4) antagonist, [Nphe(1)]nociceptin(1-13)NH(2), prevents the effects of nociceptin in neuropathic rats.

Nociceptin or orphanin FQ (N/OFQ) is the natural ligand of the opioid receptor-like 1 receptor (ORL-1), which has been also classified as the fourth member of the opioid family of receptors and named OP(4). Elucidation of the biological role of N/OFQ has been hampered by the lack of compounds that selectively block the OP(4) receptor. Recently, a N/OFQ derivative, [Nphe(1)]N/OFQ(1-13)NH(2), has been found to possess OP(4) antagonistic properties both in vitro and in vivo models. We investigated its spinal effect in the chronic constriction injury of the sciatic nerve in the rat, a model relevant to neuropathic pain in humans. Intrathecal (i.t.) administration of N/OFQ (0.2--20 nmoles) dose-dependently reversed mechanical allodynic-like behavior, while [Nphe(1)]N/OFQ(1-13)NH(2) (20--120 nmoles, i.t.) was ineffective on its own. [Nphe(1)]N/OFQ(1-13)NH(2) (60--120 nmoles, i.t.) antagonized N/OFQ (about 80% of reduction) but did not modify the activity of morphine (20 nmoles, i.t.). These results further support, for the first time in a chronic model of pain, the specific antagonistic profile of [Nphe(1)]N/OFQ(1-13)NH(2)vs the OP(4) receptor. This pseudopeptide is an interesting pharmacological tool to better clarify the role of N/OFQ in pathophysiology.

Tumor necrosis factor alpha mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity.

Severe quantitative and qualitative brown adipocyte defects are common in obesity. To investigate whether aberrant expression of tumor necrosis factor alpha (TNF-alpha) in obesity is involved in functional brown fat atrophy, we have studied genetically obese (ob/ob) mice with targeted null mutations in the genes encoding the two TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apoptosis and an increase in beta(3)-adrenoreceptor and uncoupling protein-1 expression in obese mice. Increased numbers of multilocular functionally active brown adipocytes, and improved thermoregulation was also observed in obese animals lacking TNF-alpha function. These results indicate that TNF-alpha plays an important role in multiple aspects of brown adipose tissue biology and mediates the abnormalities that occur at this site in obesity.

Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures.

1. In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures. 2. Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S-nitroso-acetyl penicillamine (SNAP) or S-nitroso-L-glutathione (GSNO). Both agents (300 microM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors. 3. Daily treatment with nitric oxide synthase inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), led to the stimulation of cell proliferation by 44+/-5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth. 4. Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L-NAME was without effect. 5. The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor-gamma and uncoupling protein-1, which are upregulated during differentiation. 6. Increasing cyclic GMP in cells by 8-bromo-cyclic GMP (100-1000 microM) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO-stimulated guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), reduced the expression of peroxisome proliferator activated receptor-gamma and uncoupling protein-1.

Advances in pharmacotherapy for obesity.

Anorectic drugs are increasingly being used in obesity to induce and/or maintain weight loss. We have focused on the development of metabolic enhancers. These compounds increase energy expenditure, which is important because weight loss is associated with metabolic re-adjustment to reduce energy output. Thus, metabolic enhancers ensure that energy expenditure is maintained when food intake is reduced. Beta3-adrenoceptor agonists are thermogenic agents that increase energy output by stimulating heat generation. Early selective beta3-agonists were effective in producing weight loss in obese rats, but were largely ineffective in humans. In addition, many interacted with other types of beta receptor to produce side-effects. The development of a Chinese hamster ovary cell (CHO) transfection system, using the human beta3-adrenoceptor gene, resulted in potential new selective human beta3-agonists being identified. However, the in vitro activity of these agents does not necessarily reflect their action in vivo, due to the presence of other receptor types and G proteins in the target cells, and interactions between them. The characterization of a selective beta3-antagonist, SR59230A, has allowed us to examine beta3-agonist activity in different experimental systems. The CHO transfection system has been used to show that SR59230A is effective in blocking agonist activity against both the rat adrenoceptor, and three human beta3-receptor isoforms. In addition, SR59230A shows competitive inhibition of agonist activity in both rat and human model systems. This antagonist may therefore provide a pharmacological tool for the functional study of by newly identified beta3-receptor agonists.

SR59230A blocks beta3-adrenoceptor-linked modulation of upcoupling protein-1 and leptin in rat brown adipocytes.

Experimental evidence suggests that, by stimulating energy expenditure in brown fat, selective beta3-adrenoceptor agonists can reduce body weight in obese rodents. In order to investigate further the physiological role of beta3-adrenoceptors in brown adipocytes, we analysed the effects of selective beta3-adrenoceptor agonists and antagonists on uncoupling protein-1 and leptin gene expression in culture-differentiated brown fat cells. Our main findings were that: (i) the leptin gene is expressed in brown adipocytes; (ii) the selective beta3-adrenoceptor agonist, N[(2S)-7-carbethoxy-1,2,3,4-tetrahydronaphth-2-yl]-(2R)-2-hydroxy- 2-(3-chlorophenil)ethanamine hydrochloride (SR58611A), inhibits leptin gene while inducing uncoupling protein-1 gene expression; (iii) these opposite effects of SR58611A are antagonized by the selective beta3-adrenoceptor antagonist, SS-enantiomer 3-(2-ethylphenoxy)-1-(1S),2,3,4-tetrahydronaphth-1-ylamin ol]-(2S)-2-propanol oxalate (SR59230A), but not by the selective beta1-adrenoceptor antagonist (+/-)-[2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4(1-methyl- 4-trifluoromethyl-2-imidazolyl)-phenoxy]-2 propanol (CGP20712A); and (iv) these effects are due to increased cyclic AMP levels. These results confirm by means of a different experimental approach that beta3-adrenoceptors play a central role in controlling the expression of genes that are important for brown fat function.

Bcl-2 and Bax are involved in the sympathetic protection of brown adipocytes from obesity-linked apoptosis.

Obesity is linked to functional brown adipose tissue (BAT) atrophy, partially due to adipocyte apoptosis. The brown adipocytes of obese rats have lower Bcl-2/Bax mRNA and protein ratios than those of their lean littermates. Exposure to a low temperature for three days markedly increased the Bcl-2/Bax ratio, by increasing the noradrenergic output to BAT, which has previously been shown to reduce apoptotic cell death. This effect could be mimicked in vitro by the addition of noradrenaline (NA) to brown adipocytes differentiated in culture. Micromolar NA concentrations increased the Bcl-2/Bax mRNA and protein ratios, and protected against serum deprivation-induced apoptosis. We conclude that NA acts by modulating bcl-2 and bax gene expression.

Inducible nitric oxide synthase in rat brown adipocytes: implications for blood flow to brown adipose tissue.

Exposure of rat brown adipocytes differentiated in culture to norepinephrine (NE) results in the production of nitrites (NO2-), the breakdown product of nitric oxide (NO). This production, which is blocked by actinomycin D1 is directly related to the duration of exposure to and dose of NE. Cytosol from NE-treated brown fat cells, but not from untreated cultures, catalyzed the Ca(2+)-independent conversion of L-arginine to L-citrulline, which could be significantly blocked by the specific nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester. Reverse transcriptase-PCR demonstrates that the addition of NE; selective beta 1-, beta 2-, or beta 3-adrenergic receptor agonists; or agents increasing cAMP production, such as forskolin, to brown adipocytes stimulates inducible NOS (iNOS) messenger RNA, which is present within 4 h after exposure. That iNOS is synthesized in brown fat cells is confirmed by immunoblotting using an antibody to the iNOS of mouse macrophages, Finally, in both brown adipose tissue (BAT) and brown adipocyte preparations from animals exposed to low temperature, iNOS messenger RNA and protein were expressed, and NOS activity was detectable; these findings were unlikely for room temperature-acclimated rats. We conclude that brown fat cells can express an inducible form of NOS similar to the iNOS of macrophages, and that its production is directly dependent on sympathetic activity in physiological conditions. NO generated by stimulation of iNOS in brown adipocytes may represent an important mechanism to modulate different BAT functions, among which is vasodilation of the BAT microcirculation.

Tumor necrosis factor-alpha induces apoptosis in rat brown adipocytes.

Accumulating evidence demonstrates that adipose tissue is a major site of tumor necrosis factor-alpha (TNF-alpha) gene expression, which is markedly high in obese animals and may contribute to obesity-linked insulin resistance. We now report that recombinant murine TNF-alpha triggers the apoptotic degeneration of brown adipocytes differentiated in culture. Moreover, noradrenaline, which has been described as having trophic effects on brown fat and accelerating the differentiation of brown adipocytes, is capable of dose-dependently preventing the TNF-alpha-induced apoptosis of brown fat cells. Since obesity is characterized by greatly increased TNF-alpha production and reduced catecholaminergic activity, apoptosis was studied in the brown fat of genetically obese animals. In situ DNA fragmentation analysis revealed a larger number of apoptotic cells in the brown fat of obese (fa/fa) than in that of lean (+/+) Zucker rats. The exposure of obese rats to low temperatures for 7 days, which increases the sympathetic activity of brown adipose tissue, significantly reduces the number of apoptotic brown adipocytes. We hypothesize that TNF-alpha may play a significant role in the control of brown fat homeostasis.

Native nicotinic acetylcholine receptors in human Imr32 neuroblastoma cells: functional, immunological and pharmacological properties.

IMR32 cells express two classes of surface nicotinic receptors: those labelled with high affinity by [125I]neuronal toxin, and those labelled by [125I]alpha-bungarotoxin. Whole-cell patch-clamp recordings indicate that both classes of receptor are able to elicit inward currents that are totally blocked by d-tubocurarine but only partially blocked by alpha-bungarotoxin. In IMR32 cells, nicotine induces an increase in the intracellular level of free Ca2+. This increase, which is also completely blocked by d-tubocurarine and only partially blocked by alpha-bungarotoxin and Cd2+, is due to extracellular calcium influx through both the nicotinic receptors and the voltage-activated Ca2+ channels. By using subunit-specific polyclonal antibodies, we have demonstrated that the alpha-bungarotoxin receptors contain the alpha 7 subunit, but none of the other subunits whose transcripts are present in IMR32 cells. The pharmacological profile of these human alpha 7-containing alpha-bungarotoxin receptors is similar to that observed in the native chick alpha 7 receptor, but there are also some species-specific differences.

Expression of alpha-bungarotoxin receptor subtypes in chick central nervous system during development.

Chick central nervous system (CNS) expresses alpha-bungarotoxin (alpha Bgtx) receptors. We have recently reported the purification and characterization of two alpha Bgtx receptor subtypes, alpha 7 and alpha 7-alpha 8 from chick optic lobe (COL). In order to study whether other alpha Bgtx receptor subtypes are present in other areas of the chick CNS, as well as their developmental expression, we used anti-alpha 7 and anti-alpha 8 subunit-specific antibodies to study alpha Bgtx receptors at different developmental stages in COL, brain and retina. We found that only the alpha 7 and alpha 7-alpha 8 subtypes are present at all developmental stages in chick COL and brain, where they represent 90% of all the alpha Bgtx receptors at embryonic day 19 and 1 day post hatching (D1). In chick retina, an alpha 8 subtype representing 50% of all alpha Bgtx receptors at D1 is present in addition to the alpha 7 and alpha 7-alpha 8 subtypes, and the expression of this alpha 8 subtype increases during neurodevelopment.

Anti-peptide specific antibodies for the characterization of different alpha subunits of alpha-bungarotoxin binding acetylcholine receptors present in chick optic lobe.

Chick optic lobe express alpha-Bungarotoxin receptors. We have recently purified these receptors which, when reconstituted in a lipid bilayer, behave as functional acetylcholine gated channels. In order to characterize this purified preparation, we raised polyclonal antibodies against peptides obtained from the putative cytoplasmic domain between the hydrophobic sequence M3 and M4 of two previously cloned alpha-Bungarotoxin receptor subunits, alpha 7 and alpha 8. Both antibodies recognized the receptors present in the membrane extract and in the purified preparation, although the amount of the alpha-Bungarotoxin receptors precipitated by the two antibodies was quantitatively different. In Western blots of both purified and membrane-bound receptors, these antibodies specifically reacted with an M(r) 57000-55000 band. A study was also undertaken to quantify the receptors containing these subunits in different chick brain areas; it was found that the number of these subunits, as well as their ratio, was similar in all the tested areas. Furthermore, the alpha-Bungarotoxin receptors were present in at least two subtypes, one containing only the alpha 7 subunit and the other both alpha 7 and alpha 8 subunits.

A functional alpha-bungarotoxin receptor is present in chick cerebellum: purification and characterization.

It has recently been demonstrated that alpha-bungarotoxin receptors, which behave as functional nicotinic receptors, are present in chick CNS. In this paper, we report the purification and characterization of a functional alpha-bungarotoxin receptor from chick cerebellum, a nervous tissue in which a clear inhibition of induced nicotine effects has been reported in vivo. This receptor contains at least three subunits of apparent mol. wt 52,000, 57,000 and 67,000. The use of monoclonal antibodies specific for the alpha 7 subunit demonstrated that 75% of the molecules present in our purified preparation belong to the alpha 7 subtype and that this antibody labels the 57,000 band in western blot, thus indicating that this is the toxin binding subunit. Reconstruction experiments in planar lipid bilayers show that this alpha-bungarotoxin receptor forms a cation selective channel whose opening is blocked by d-tubocurarine. Binding experiments on immobilized receptors over an alpha-bungarotoxin-Sepharose affinity column show that the ligand binding subunit is present in vivo in two copies per receptor. Immunological, pharmacological and functional experiments show that this purified receptor is very similar, but not identical, to the previously characterized chick optic lobe receptor, thus indicating the heterogeneity of these alpha-bungarotoxin receptors in the CNS.