Biomedical subjects

Saverio Cinti

Publications and source records attributed to Saverio Cinti.

34 records · Page 2Linked to original sources

PubMed2004

The imprinted signaling protein XL alpha s is required for postnatal adaptation to feeding.

Genomic imprinting, by which maternal and paternal alleles of some genes have different levels of activity, has profound effects on growth and development of the mammalian fetus. The action of imprinted genes after birth, in particular while the infant is dependent on maternal provision of nutrients, is far less well understood. We disrupted a paternally expressed transcript at the Gnas locus, Gnasxl, which encodes the unusual Gs alpha isoform XL alpha s. Mice with mutations in Gnasxl have poor postnatal growth and survival and a spectrum of phenotypic effects that indicate that XL alpha s controls a number of key postnatal physiological adaptations, including suckling, blood glucose and energy homeostasis. Increased cAMP levels in brown adipose tissue of Gnasxl mutants and phenotypic comparison with Gnas mutants suggest that XL alpha s can antagonize Gs alpha-dependent signaling pathways. The opposing effects of maternally and paternally expressed products of the Gnas locus provide tangible molecular support for the parental-conflict hypothesis of imprinting.

Adipose Tissue↗

PubMed2004

Retinoblastoma protein functions as a molecular switch determining white versus brown adipocyte differentiation.

Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma gene-deficient (Rb-/-) MEFs and stem cells, but not the corresponding wild-type cells, differentiate into adipocytes with a gene expression pattern and mitochondria content resembling brown adipose tissue. pRB-deficient MEFs exhibit an increased expression of the Forkhead transcription factor Foxc2 and its target gene cAMP-dependent protein kinase regulatory subunit RIalpha, resulting in increased cAMP sensitivity. Suppression of cAMP-dependent protein kinase activity in Rb(-/-)MEFs blocked the brown adipocyte-like gene expression pattern without affecting differentiation per se. Immunohistochemical studies revealed that pRB is present in the nuclei of white but not brown adipocyte precursor cells at a developmental stage where both cell types begin to accumulate lipid and brown adipocytes express UCP-1. Furthermore, pRB rapidly undergoes phosphorylation upon cold-induced neodifferentiation and up-regulation of UCP-1 expression in brown adipose tissue. Finally, down-regulation of pRB expression accompanies transdifferentiation of white into brown adipocytes in response to beta3-adrenergic receptor agonist treatment. We propose that pRB acts as a molecular switch determining white vs. brown adipogenesis, suggesting a previously uncharacterized function of this key cell cycle regulator in adipocyte lineage commitment and differentiation.

Adipocytes↗

PubMed2004

Zinc-alpha2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia.

Zinc-alpha2-glycoprotein (ZAG), a 43-kDa protein, is overexpressed in certain human malignant tumors and acts as a lipid-mobilizing factor to stimulate lipolysis in adipocytes leading to cachexia in mice implanted with ZAG-producing tumors. Because white adipose tissue (WAT) is an endocrine organ secreting a wide range of protein factors, including those involved in lipid metabolism, we have investigated whether ZAG is produced locally by adipocytes. ZAG mRNA was detected by RT-PCR in the mouse WAT depots examined (epididymal, perirenal, s.c., and mammary gland) and in interscapular brown fat. In WAT, ZAG gene expression was evident in mature adipocytes and in stromal-vascular cells. Using a ZAG Ab, ZAG protein was located in WAT by Western blotting and immunohistochemistry. Mice bearing the MAC16-tumor displayed substantial losses of body weight and fat mass, which was accompanied by major increases in ZAG mRNA and protein levels in WAT and brown fat. ZAG mRNA was detected in 3T3-L1 cells, before and after the induction of differentiation, with the level increasing progressively after differentiation with a peak at days 8-10. Both dexamethasone and a beta3 agonist, BRL 37344, increased ZAG mRNA levels in 3T3-L1 adipocytes. ZAG gene expression and protein were also detected in human adipose tissue (visceral and s.c.). It is suggested that ZAG is a new adipose tissue protein factor, which may be involved in the modulation of lipolysis in adipocytes. Overexpression in WAT of tumor-bearing mice suggests a local role for adipocyte-derived ZAG in the substantial reduction of adiposity of cancer cachexia.

3T3 Cells↗

PubMed2004

Mosaic analysis of insulin receptor function.

Insulin promotes both metabolism and growth. However, it is unclear whether insulin-dependent growth is merely a result of its metabolic actions. Targeted ablation of insulin receptor (Insr) has not clarified this issue, because of early postnatal lethality. To examine this question, we generated mice with variable cellular mosaicism for null Insr alleles. Insr ablation in approximately 80% of cells caused extreme growth retardation, lipoatrophy, and hypoglycemia, a clinical constellation that resembles the human syndrome of leprechaunism. Insr ablation in 98% of cells, while resulting in similar growth retardation and lipoatrophy, caused diabetes without beta-cell hyperplasia. The growth retardation was associated with a greater than 60-fold increase in the expression of hepatic insulin-like growth factor binding protein-1. These findings indicate that insulin regulates growth independently of metabolism and that the number of insulin receptors is an important determinant of the specificity of insulin action.

Adipose Tissue↗

PubMed2004

Presence and distribution of cholinergic nerves in rat mediastinal brown adipose tissue.

Brown adipose tissue (BAT) is richly provided with sympathetic noradrenergic nerves but is believed to lack a parasympathetic nerve supply. Acetylcholine is the predominant transmitter of postganglionic parasympathetic nerves. The vesicular acetylcholine transporter (VAChT) resides in synaptic vesicles of cholinergic nerve terminals and is used as a marker for peripheral cholinergic nerves. We sought cholinergic nerves in rat BAT using VAChT immunohistochemistry (IHC) on cryosections of interscapular, cervical, mediastinal, and perirenal depots. Mediastinal BAT was the sole depot provided with putative parasympathetic perivascular and parenchymal cholinergic nerves. The absence of vasoactive intestinal peptide-positive nerves suggested their nature as pure cholinergic fibers. By confocal microscopy, both cholinergic and noradrenergic nerves were detected in mediastinal BAT. Cold exposure and fasting led to increased density of VAChT-positive fibers and of noradrenergic sympathetic nerves at morphometry. The unexpected double innervation of mediastinal BAT may explain the inhibitory influence on thermogenesis observed after systemic injection of muscarinic antagonists in rats, and raises questions about the physiological role of its cholinergic nerve supply.

Acetylcholine↗

PubMed2003

Sema3A and neuropilin-1 expression and distribution in rat white adipose tissue.

Semaphorins are cell surface and/or soluble signals that exert an inhibitory control on axon guidance. Sema3A, the vertebrate-secreted semaphorin, binds to neuropilin-1, which together with plexins constitutes the functional receptor. To verify whether Sema3A is produced by white adipocytes and, in that case, to detect its targets in white adipose tissue, we studied the cell production and tissue distribution of Sema3A and neuropilin-1 in rat retroperitoneal and epididymal adipose depots. Sema3A and neuropilin-1 were detected in these depots by Western blotting. The immunohistochemical results showed that Sema3A is produced in, and possibly secreted by, smooth muscle cells of arteries and white adipocytes. Accordingly, neuropilin-1 was found on perivascular and parenchymal nerves. Such a pattern of distribution is in line with a role for secreted Sema3A in the growth and plasticity of white adipose tissue nerves. Indeed, after fasting, when white adipocytes are believed to be overstimulated by noradrenaline and rearrangement of the parenchymal nerve supply may occur, adipocytic expression of Sema3A is reduced. Finally, the presence of neuropilin-1 in some white adipocytes raises the interesting possibility that Sema3A also exerts an autocrine-paracrine role on these cells.

Adipocytes↗

PubMed2003

Beta 3-adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat.

White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by beta-adrenoceptors but there is a controversy about the subtype(s) involved. In the present study, we exposed to cold beta 3-adrenoceptor knockout mice (beta 3KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein-1 (UCP1). In brown fat of beta 3KO mice, UCP1 expression levels were normal at 24 degrees C as well as after a 10-day cold exposure. Strikingly, under both conditions, in the white fat of beta 3KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that beta 3-adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat.

Adipocytes↗

PubMed2003

Human metabolic syndrome resulting from dominant-negative mutations in the nuclear receptor peroxisome proliferator-activated receptor-gamma.

We previously reported a syndrome of severe hyperinsulinemia and early-onset hypertension in three patients with dominant-negative mutations in the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-gamma. We now report the results of further detailed pathophysiological evaluation of these subjects, the identification of affected prepubertal children within one of the original families, and the effects of thiazolidinedione therapy in two subjects. These studies 1) definitively demonstrate the presence of severe peripheral and hepatic insulin resistance in the affected subjects; 2) describe a stereotyped pattern of partial lipodystrophy associated with all the features of the metabolic syndrome and nonalcoholic steatohepatitis; 3) document abnormalities in the in vivo function of remaining adipose tissue, including the inability of subcutaneous abdominal adipose tissue to trap and store free fatty acids postprandially and the presence of very low circulating levels of adiponectin; 4) document the presence of severe hyperinsulinemia in prepubertal carriers of the proline-467-leucine (P467L) PPAR-gamma mutation; 5) provide the first direct evidence of cellular resistance to PPAR-gamma agonists in mononuclear cells derived from the patients; and 6) report on the metabolic response to thiazolidinedione therapy in two affected subjects. Although the condition is rare, the study of humans with dominant-negative mutations in PPAR-gamma can provide important insight into the roles of this nuclear receptor in human metabolism.

Abdomen↗

PubMed2002

betaAR signaling required for diet-induced thermogenesis and obesity resistance.

Excessive caloric intake is thought to be sensed by the brain, which then activates thermogenesis as a means of preventing obesity. The sympathetic nervous system, through beta-adrenergic receptor (betaAR) action on target tissues, is likely the efferent arm of this homeostatic mechanism. To test this hypothesis, we created mice that lack the three known betaARs (beta-less mice). beta-less mice on a Chow diet had a reduced metabolic rate and were slightly obese. On a high-fat diet, beta-less mice, in contrast to wild-type mice, developed massive obesity that was due entirely to a failure of diet-induced thermogenesis. These findings establish that betaARs are necessary for diet-induced thermogenesis and that this efferent pathway plays a critical role in the body's defense against diet-induced obesity.

Adipose Tissue, Brown↗

PubMed2002

Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus.

The intracellular localization and activity of the nitric oxide synthase (NOS) isoforms were investigated in rat brown adipocytes. Immunohistochemistry showed cytoplasmic and nuclear staining for the endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms; accordingly, anti-L-citrulline antibody, a marker of NOS activity, immunostained both the cytoplasm and the nucleus. The presence of metabolically active NOS in the nucleus was further confirmed by immunoblotting analyses of subcellular fractions of homogenates from cultured brown adipocytes and by measurements of NOS activity in the cytosol and nucleus. Sympathetic stimulation in vivo (i.e. cold exposure or beta(3)-adrenergic agonist treatment) and in vitro (i.e. noradrenaline treatment of cultured cells) significantly increased both cytosolic and nuclear eNOS and iNOS expression and activities. By contrast, the number of iNOS-positive, but not eNOS-positive, nuclei was significantly lower in the functionally impaired brown fat of genetically obese Zucker fa/fa rats. These data suggest the existence of a noradrenaline-modulated functional NOS system in the nucleus of brown adipocytes.

Adipocytes↗

PubMed2002

Perinatal expression of leptin in rat stomach.

It has been reported recently that the stomach can produce and store leptin and release it, both into the blood and into the gastrointestinal lumen, in response to food intake. Here, we have followed the ontogenic pattern of leptin mRNA expression and leptin levels in stomach during the perinatal period, which were compared with adults. Leptin mRNA expression was assessed by reverse transcriptase-polymerase chain reaction, and tissue leptin content by enzyme-linked immunosorbent assay and localised by immunohistochemistry. Leptin mRNA is expressed at low levels in rat stomach in prenatal stages. It increased from 4 to 8 hr of life in suckling rats, an increase not observed in the fasted pups, which were separated from their mothers immediately after birth. Leptin expression rose steadily after birth during the first month of life, with a marked increase from 15-day-old rats, followed by a parallel increase in leptin levels from day 21 of life, which was coincident with the change from suckling to a solid diet. The immunohistochemical analysis showed leptin immunoreactivity at different levels of the stomach mucosa, suggesting that during early development leptin could derive from different sources. During the pre- and neonatal periods, leptin is mainly located at the superficial epithelium (suggesting maternal origin from amniotic cells and mammary glandular cells, respectively). At the beginning of the chow diet, the stomach produces leptin in the glands (main source from 15 days of life), suggesting an endogenous production of the protein after that period. The present work demonstrates the expression of leptin mRNA and leptin protein in the stomach of neonate rats, and shows that the ontogenic profile of leptin appearance in the stomach during the perinatal period is probably related to the onset of suckling and to the change of diet from milk to solid chow.

Animals↗

PubMed2002

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.

Adipocytes↗

PubMed2002

Expression of the uncoupling protein 1 from the aP2 gene promoter stimulates mitochondrial biogenesis in unilocular adipocytes in vivo.

Mitochondrial uncoupling protein 1 (UCP1) is a specific marker of multilocular brown adipocytes. Ectopic UCP1 in white fat of aP2-Ucp1 mice mitigates development of obesity by both, increasing energy expenditure and decreasing in situ lipogenesis. In order to further analyse consequences of respiratory uncoupling in white fat, the effects of the ectopic UCP1 on the morphology of adipocytes and biogenesis of mitochondria in these cells were studied. In subcutaneous white fat of both aP2-Ucp1 and young control (5-week-old) mice, numerous multilocular adipocytes were found, while they were absent in adult (7- to 9-month-old) animals. Only unilocular cells were present in epididymal fat of both genotypes. In both fat depots of aP2-Ucp1 mice, the levels of the UCP1 transcript and UCP1 antigen declined during ageing, and they were higher in subcutaneous than in epididymal fat. Under no circumstances could ectopic UCP1 induce the conversion of unilocular into multilocular adipocytes. Presence of ectopic UCP1 in unilocular adipocytes was associated with the elevation of the transcripts for UCP2 and for subunit IV of mitochondrial cytochrome oxidase (COX IV), and increased content of mitochondrial cytochromes. Electron microscopy indicated changes of mitochondrial morphology and increased mitochondrial content due to ectopic UCP1 in unilocular adipocytes. In 3T3-L1 adipocytes, 2,4-dinitrophenol increased the levels of the transcripts for both COX IV and for nuclear respiratory factor-1. Our results indicate that respiratory uncoupling in unilocular adipocytes of white fat is capable of both inducing mitochondrial biogenesis and reducing development of obesity.

Adipocytes↗

PubMed2002

Defective insulin secretion in pancreatic beta cells lacking type 1 IGF receptor.

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of beta cell mass and impaired glucose-dependent insulin release. beta cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in beta cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect beta cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion.

Animals↗

PubMed2002

CL316,243 and cold stress induce heterogeneous expression of UCP1 mRNA and protein in rodent brown adipocytes.

Uncoupling protein 1 (UCP1), the mammalian thermogenic mitochondrial protein, is found only in brown adipocytes, but its expression by immunohistochemistry is not homogeneous. Here we present evidence that the non-homogeneous pattern of immunostaining for UCP1 (referred to as the "Harlequin phenomenon") is particularly evident after acute and chronic cold (4C) stimulus and after administration of a specific beta(3)-adrenoceptor agonist (CL316,243). Accordingly, mRNA in situ expression confirmed the UCP1 non-homogeneous pattern of gene activation under conditions of adrenergic stimulus. Furthermore, morphometric analysis of immunogold-stained thin sections showed that UCP1-gold particle density was different among neighboring brown adipocytes with mitochondria of the same size and density. When the adrenergic stimulus was reduced in warm-acclimated animals (28C), UCP1 protein and mRNA expression was reduced and consequently the Harlequin phenomenon was barely visible. These data suggest the existence of an alternative and controlled functional recruitment of brown adipocytes in acute adrenergically stressed animals, possibly to avoid heat and metabolic damage in thermogenically active cells. Of note, the heat shock protein heme oxygenase 1 (HO1) is heterogeneously expressed in adrenergically stimulated brown adipose tissue and, specifically, cells expressing strong immunoreactivity for UCP1 also strongly express HO1.

Acclimatization↗

PubMed2002

Insulin resistance in morbid obesity: reversal with intramyocellular fat depletion.

Obesity is a frequent cause of insulin resistance and poses a major risk for diabetes. Abnormal fat deposition within skeletal muscle has been identified as a mechanism of obesity-associated insulin resistance. We tested the hypothesis that dietary lipid deprivation may selectively deplete intramyocellular lipids, thereby reversing insulin resistance. Whole-body insulin sensitivity (by the insulin clamp technique), intramyocellular lipids (by quantitative histochemistry on quadriceps muscle biopsies), muscle insulin action (as the expression of Glut4 glucose transporters), and postprandial lipemia were measured in 20 morbidly obese patients (BMI = 49 +/- 8 [mean +/- SD] kg x m(-2)) and 7 nonobese control subjects. Patients were restudied 6 months later after biliopancreatic diversion (BPD; n = 8), an operation that induces predominant lipid malabsorption, or hypocaloric diet (n = 9). At 6 months, BPD had caused the loss of 33 +/- 10 kg through lipid malabsorption (documented by a flat postprandial triglyceride profile). Despite an attained BMI still in the obese range (39 +/- 8 kg x m(-2)), insulin resistance (23 +/- 3 micromol/min per kg of fat-free mass; P < 0.001 vs. 53 +/- 13 of control subjects) was fully reversed (52 +/- 11 micromol/min per kg of fat-free mass; NS versus control subjects). In parallel with this change, intramyocellular-but not perivascular or interfibrillar-lipid accumulation decreased (1.63 +/- 1.06 to 0.22 +/- 0.44 score units; P < 0.01; NS vs. 0.07 +/- 0.19 of control subjects), Glut4 expression was restored, and circulating leptin concentrations were normalized. In the diet group, a weight loss of 14 +/- 12 kg was accompanied by very modest changes in insulin sensitivity and intramyocellular lipid contents. We conclude that lipid deprivation selectively depletes intramyocellular lipid stores and induces a normal metabolic state (in terms of insulin-mediated whole-body glucose disposal, intracellular insulin signaling, and circulating leptin levels) despite a persistent excess of total body fat mass.

Adult↗