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Biomedical subjects

E Nisoli

Publications and source records attributed to E Nisoli.

At least 19 recordsLinked to original sources
PubMed2001

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.

Adipose Tissue, Brown↗
PubMed2001

Efficacy and tolerability of moclobemide in bulimia nervosa: a placebo-controlled trial.

A 6-week double-blind placebo-controlled trial was carried out to examine the efficacy and tolerability of moclobemide, a monoamine oxidase type A selective and reversible inhibitor, in the treatment of bulimia nervosa. Patients were admitted to the study even if they were unable to adhere to a tyramine-free diet. Fifty-two normal-weight women (age range 18-40 years) suffering from bulimia nervosa (DSM-IV criteria) completed the trial. Particular emphasis was placed on evaluating the incidence of hypertension and other side-effects in chronically treated patients. At the usual antidepressant dose of 600 mg, moclobemide was not significantly superior to placebo in the reducing the weekly number of binge eating episodes or in improving several measures of eating attitudes and behaviour (BITE, EDI, TFEQ) in normal-weight bulimia nervosa. The dropout rate was relatively low (29%), and the side-effects were limited and equally distributed between the two treatment groups. No patient experienced a hypertensive crisis during the study and no serious side-effect was detected. The study indicates that moclobemide 600 mg pro die is not efficacious in bulimia nervosa, but it can be safely administered, even to young subjects, at a very high risk of consuming large amounts of tyramine-rich foods without dietary restrictions.

Adolescent↗
PubMed2001

Preferential channeling of energy fuels toward fat rather than muscle during high free fatty acid availability in rats.

The preferential channeling of different fuels to fat and changes in the transcription profile of adipose tissue and skeletal muscle are poorly understood processes involved in the pathogenesis of obesity and insulin resistance. Carbohydrate and lipid metabolism may play relevant roles in this context. Freely moving lean Zucker rats received 3- and 24-h infusions of Intralipid (Pharmacia and Upjohn, Milan, Italy) plus heparin, or saline plus heparin, to evaluate how an increase in free fatty acids (nonesterified fatty acid [NEFA]) modulates fat tissue and skeletal muscle gene expression and thus influences fuel partitioning. Glucose uptake was determined in various tissues at the end of the infusion period by means of the 2-deoxy-[1-3H]-D-glucose technique after a euglycemic-hyperinsulinemic clamp: high NEFA levels markedly decreased insulin-mediated glucose uptake in red fiber-type muscles but enhanced glucose utilization in visceral fat. Using reverse transcriptase-polymerase chain reaction and Northern blotting analyses, the mRNA expression of fatty acid translocase (FAT)/CD36, GLUT4, tumor necrosis factor (TNF)-alpha, peroxisome proliferator-activated receptor (PPAR)-gamma, leptin, uncoupling protein (UCP)-2, and UCP-3 was investigated in different fat depots and skeletal muscles before and after the study infusions. GLUT4 mRNA levels significantly decreased (by approximately 25%) in red fiber-type muscle (soleus) and increased (by approximately 45%) in visceral adipose tissue. Furthermore, there were marked increases in FAT/CD36, TNF-alpha, PPAR-gamma, leptin, UCP2, and UCP3 mRNA levels in the visceral fat and muscle of the treated animals in comparison with those measured in the saline-treated animals. These data suggest that the in vivo gene expression of FAT/CD36, GLUT4, TNF-alpha, PPAR-gamma, leptin, UCP2, and UCP3 in visceral fat and red fiber-type muscle are differently regulated by circulating lipids and that selective insulin resistance seems to favor, at least in part, a prevention of fat accumulation in tissues not primarily destined for fat storage, thus contributing to increased adiposity and the development of a prediabetic syndrome.

Adipose Tissue↗
PubMed2000

Expression and distribution of heme oxygenase-1 and -2 in rat brown adipose tissue: the modulatory role of the noradrenergic system.

To investigate whether brown adipose tissue (BAT) expresses the inducible (HO-1) and the constitutive (HO-2) isoform of heme oxygenase, reverse transcriptase-polymerase chain reaction, Western blotting and immunohistochemistry were performed on interscapular BAT (IBAT) from rats acclimated at environmental temperature or exposed to cold. Both HO isoforms were detected in rat IBAT. They were immunolocalized in the cytoplasm and/or nuclei of brown adipocytes, in parenchymal capillaries, arteries and in some veins and nerves. Whereas cold exposure did not affect HO-2 expression, it significantly increased the expression of HO-1, both at mRNA (about 3-fold) and protein (about 2-fold) levels, reflecting the increased expression of HO-1 in the brown adipocytes and endothelial cells of parenchymal capillaries. Western blotting of cytosolic and nuclear protein extracts from cultured differentiated brown adipocytes showed that HO-1 and HO-2 are indeed localized in the cytosol and nuclei of brown adipocytes, and that noradrenaline stimulation significantly increased their amount in cytosol but not in the nuclear fraction.

Acclimatization↗
PubMed2000

Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells.

In previous studies we have showed that somatostatin (SST) inhibits cell division, mitogen-activated protein (MAP) kinase and Ras activity in the human neuroblastoma cell line SY5Y. In the present study, we have assessed the role of a series of SST analogs, three of which were selective for SSTR1, SSTR2 or SSTR5, in these cellular events. All the analogs inhibited forskolin-induced cAMP accumulation. Selective stimulation of SSTR1 or SSTR2 but not of SSTR5 inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation. The three analogs inhibited PDGF-stimulated MAP kinase activity, at least at an early time. In contrast, none of the analogs used individually was able to inhibit PDGF-stimulated Ras activity. A combined stimulation of SSTR2 and SSTR5 was necessary to obtain a significant inhibitory effect, suggesting the possibility of receptor heterodimerization. These results indicate that SST inhibition of Ras and MAP kinase activities takes place via different pathways and that SST inhibition of PDGF-induced cell proliferation occurs via a Ras-independent pathway.

Animals↗
PubMed2000

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.

Adaptation, Physiological↗
PubMed2000

Induction of fatty acid translocase/CD36, peroxisome proliferator-activated receptor-gamma2, leptin, uncoupling proteins 2 and 3, and tumor necrosis factor-alpha gene expression in human subcutaneous fat by lipid infusion.

Little is known about the mechanisms involved in the preferential channeling of different fuels to fat and how the target tissue participates in this process. Dietary fatty acids have been shown to act as signaling molecules that bind and activate a new class of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs). PPAR-gamma is particularly interesting because it may have the potential to link particular fatty acids with a program of gene expression involved in lipid storage and metabolism. We investigated whether a nutrient-sensing pathway is activated by an increased availability of lipid fuels in nine normal weight male volunteers. Using reverse transcriptase-polymerase chain reaction analysis, the mRNA expression of fatty acid translocase (FAT)/CD36, PPAR-gamma2, leptin, uncoupling protein (UCP)-2 and UCP-3, and tumor necrosis factor (TNF)-alpha was investigated in gluteal subcutaneous fat biopsies before and after 5 h infusions of saline or Intralipid (Pharmacia and Upjohn, Milan, Italy) plus heparin, which does not modify insulinemia. Marked increases in FAT/CD36 (724+/-18%; P < 0.05), PPAR-gamma2 (200+/-8%; P < 0.05), leptin (110+/-13%; P < 0.05), UCP-2 (120+/-7%; P < 0.05), UCP-3 (80+/-5%; P < 0.05), and TNF-alpha mRNA (130+/-12%; P < 0.05) were observed in comparison with pretreatment levels, whereas there was no change after saline infusion. These data suggest that the in vivo gene expression of FAT/CD36, PPAR-gamma2, leptin, UCP-2, UCP-3, and TNF-alpha in subcutaneous adipose tissue is regulated by circulating lipids independent of insulin and that prolonged hyperlipidemia may therefore contribute to increased fat metabolism and storage as a result of the increased expression of these proteins.

Adipose Tissue↗
PubMed1999

Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats.

The thermogenic activity of brown adipose tissue (BAT) is heavily dependent on high perfusion, through its dense vascular system. Angiogenesis must go hand-in-hand with BAT functions, but little is known about the factors controlling it. In the present study we demonstrate that: (a) vascular endothelial growth factor (VEGF) is synthesised and released in brown adipocytes in culture; (b) VEGF mRNA isoforms and protein appear in dispersed mature brown adipocytes and whole tissue; (c) VEGF expression is increased in BAT from cold-exposed rats, and in cultured brown adipocytes exposed to noradrenaline and the beta3-adrenoceptor agonists; (e) BAT from genetically obese (falfa) rats exhibits reduced expression of VEGF as well as a change in the ratio of mRNA isoforms. It is concluded that sympathetic control of VEGF expression via noradrenaline acting on beta3-adrenoceptors plays a major role in developmental and adaptive angiogenesis, and defects in this contribute to the reduced thermogenic capacity of BAT in genetic obesity.

Adipose Tissue, Brown↗
PubMed1998

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.

Adipose Tissue, Brown↗
PubMed1998

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.

Adipocytes↗
PubMed1998

Nerve growth factor, beta3-adrenoceptor and uncoupling protein 1 expression in rat brown fat during postnatal development.

An analysis was made of the expression of nerve growth factor (NGF) mRNA and protein in the brown fat of rats at different ages, and the results compared with the expression of beta3-adrenoceptor and uncoupling protein 1 (UCP1). NGF, beta3-adrenoceptor, and UCP1 messenger RNA and protein levels were measured by means of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting in the brown fat of rats at different ages (from 20-day-old fetuses (E20) to 16-month-old rats). During the perinatal period, NGF production increased and then declined to adult levels (which are comparable with fetal levels) by eight months, and remained stable thereafter. Relatively low levels of NGF were present in the brown fat of aged rats. Taken together, these results suggest that NGF may be responsible for regulating sympathetic innervation during the perinatal and adult periods.

Adipose Tissue, Brown↗
PubMed1998

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.

Adipose Tissue, Brown↗
PubMed1998

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.

Adrenergic beta-Agonists↗
PubMed1997

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.

Adipocytes↗