Antonella Puglianiello

Insulin-Like Growth Factor-I and -II Levels Are Associated with the Progression of Nonalcoholic Fatty Liver Disease in Obese Children

OBJECTIVE To correlate circulating levels of insulin-like growth factor (IGF)-I, IGF-II, and IGF binding protein (IGFBP)-3 in a population of obese children with biopsy-proven nonalcoholic fatty liver disease (NAFLD) with clinical, biochemical, and histological features. STUDY DESIGN We conducted a cross-sectional study at the Hepatometabolic Unit of the Bambino Gesù Childrens Hospital, Rome, Italy. Obese children (42 girls and 57 boys) underwent liver biopsy, anthropometry, biochemical assessment, and IGF system evaluation. Serum concentrations of IGF-I, IGF-II, and IGFBP-3 were measured. The liver biopsy features of each case were graded according to the NAFLD Activity Scoring system. The degrees of steatosis, inflammation, ballooning, and fibrosis were calculated. RESULTS Nonalcoholic steatohepatitis was diagnosed in 14/99 obese subjects. Stepwise regression analysis revealed that IGF-I was the major predictor of ballooning (β = -0.463; P < .0001) and NAFLD activity score (β = -0.457; P < .0001), IGF-I/IGFBP-3 ratio was the major predictor of liver inflammation (β = -0.285; P = .005), and IGF-II was the major predictor of liver fibrosis (β = 0.343; P < .005). CONCLUSION Circulating levels of IGF-I and IGF-II are associated with the histological stages of NAFLD and may represent novel markers of liver damage progression in obese children.

Antiviral activity of individual versus combined treatments with interferon α, β and γ on early infection with HTLV-I in vitro

We have shown previously that infection of mononuclear cells derived from neonatal cord (CBMC) or adult peripheral (PBMC) blood with HTLV-1 can be controlled in vitro by treatment with interferon (IFN) alpha, beta or gamma. The activity of IFNs was mainly related to the induction of an active antiviral competence in hosts immune effector cells. The antiviral activity of IFN-boosted CBMC could be ascribed both to a positive regulation of cell-mediated immunity and to inhibition of viral infection. Data described herein provide further information on the mechanisms of the antiviral activity of IFNs and compare the activity of each type of IFN with the association of alpha + beta, alpha + gamma and beta + gamma IFNs, at a concentration of 100 or 1000 IU/ml. When added at the onset of the co-culture of CBMC with lethally irradiated, virus-donor MT-2 cells, IFNs could protect host CBMC by inhibiting HTLV-1 infection in terms of reduced proviral integration and a lower percentage of virus-positive cells, until 4 weeks of culture. Infection of CBMC was inhibited at a comparable extent by either individual or combined IFN treatments. However, a clearcut inhibition of HTLV-I transcription was found only when alpha 100 + beta 1000 IU/ml and especially alpha 1000 + gamma 100 IU/ml combined treatments were tested. When the chronically infected, virus-producing MT-2 cells were treated with IFNs, a remarkable inhibition of HTLV-I transcription was found only after multiple treatments. However, MT-2 cells became resistant to the antiviral activity of IFN gamma, but not to that of IFN alpha or beta. These data provide further information on the control of HTLV-I replication mediated by IFNs at different steps of the viral life cycle, being therefore relevant to the clinical use of combined IFNs in the treatment of acute infection. Moreover, IFNs could be used to prevent the establishment of a persistent infection, which is a prerequisite for developing adult T-cell leukemia (ATL) and/or virus-associated myelopathy.

IGF2 Methylation Is Associated with Lipid Profile in Obese Children

Aim: Our aim was to investigate the relationships between the degree of IGF2 methylation and the metabolic status in obese children and adolescents. Subjects and Methods: Eighty-five obese subjects aged 11.6 ± 2.1 years were studied. Anthropometry, metabolic parameters, blood pressure and body composition were assessed. DNA methylation analysis was performed by restriction enzyme digestion assay. The study population was subdivided into two groups according to the percentage of IGF2 cytidine-guanosine (CpG) island methylation. Results: Twenty-two subjects showed intermediate methylation (a percentage of CpG site methylation comprised between 10 and 60%), 56 were hypomethylated (percentage of methylation lower than 10%), and only 1 showed a high rate of hypermethylation (percentage of methylation above 60%). Children with intermediate methylation showed significantly higher levels of triglycerides (107.6 ± 41.99 vs. 76.6 ± 30.18 mg/dl, p < 0.005) and a higher triglyceride/high-density lipoprotein-cholesterol ratio (2.23 ± 0.98 vs. 1.79 ± 0.98, p < 0.02) compared with hypomethylated children. Conclusions: These preliminary findings show for the first time a relationship between IGF2 methylation pattern and lipid profile in obese children. Although the correlation does not imply causation, if our findings are confirmed in further studies, IGF2 methylation might represent an epigenetic marker of metabolic risk.

Uteroplacental insufficiency down regulates insulin receptor and affects expression of key enzymes of long-chain fatty acid (LCFA) metabolism in skeletal muscle at birth

BackgroundEpidemiological studies have revealed a relationship between early growth restriction and the subsequent development of insulin resistance and type 2 diabetes. Ligation of the uterine arteries in rats mimics uteroplacental insufficiency and serves as a model of intrauterine growth restriction (IUGR) and subsequent developmental programming of impaired glucose tolerance, hyperinsulinemia and adiposity in the offspring. The objective of this study was to investigate the effects of uterine artery ligation on the skeletal muscle expression of insulin receptor and key enzymes of LCFA metabolism.MethodsBilateral uterine artery ligation was performed on day 19 of gestation in Sprague-Dawley pregnant rats. Muscle of the posterior limb was dissected at birth and processed by real-time RT-PCR to analyze the expression of insulin receptor, ACCα, ACCβ (acetyl-CoA carboxylase alpha and beta subunits), ACS (acyl-CoA synthase), AMPK (AMP-activated protein kinase, alpha2 catalytic subunit), CPT1B (carnitine palmitoyltransferase-1 beta subunit), MCD (malonyl-CoA decarboxylase) in 14 sham and 8 IUGR pups.Muscle tissue was treated with lysis buffer and Western immunoblotting was performed to assay the protein content of insulin receptor and ACC.ResultsA significant down regulation of insulin receptor protein (p < 0.05) and reduced expression of ACS and ACCα mRNA (p < 0.05) were observed in skeletal muscle of IUGR newborns. Immunoblotting showed no significant change in ACCα content.ConclusionOur data suggest that uteroplacental insufficiency may affect skeletal muscle metabolism down regulating insulin receptor and reducing the expression of key enzymes involved in LCFA formation and oxidation.

Changes in the Expression of Hypothalamic Lipid Sensing Genes in Rat Model of Intrauterine Growth Retardation (IUGR).

Intrauterine growth retardation (IUGR) has been linked to the development of type 2 diabetes in later life. The mechanisms underlying this phenomenon are unknown. Recent data suggest that some of the molecular defects underlying type 2 diabetes reside in the CNS. The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where LCFA undergo β-oxidation. Hypothalamic inhibition of CPT1 decreases food intake and suppresses endogenous glucose production. Our aim was to investigate the effects of uterine artery ligation, a procedure that mimics uteroplacental insufficiency, on the CNS expression of CPT1 and other key enzymes of LCFA metabolism. Bilateral uterine artery ligation was performed on d 19 of gestation in the pregnant rat; sham-operated pregnant rats served as controls. Hypothalamus, cerebellum, hippocampus, and cortex were dissected and analyzed at birth by real-time PCR. Nonesterified fatty acid (NEFA) serum levels were significantly higher in IUGR pups (p < 0.0001). In IUGR rats, the hypothalamic expression of CPT1 isoform C (p = 0.005) and acetyl-CoA carboxylase (ACC) isoforms alpha (p < 0.05) and beta (p = 0.005) were significantly decreased. The data presented here support the hypothesis that an abnormal intrauterine milieu can induce changes in hypothalamic lipid sensing.

Exposure to Uteroplacental Insufficiency Reduces the Expression of Signal Transducer and Activator of Transcription 3 and Proopiomelanocortin in the Hypothalamus of Newborn Rats

IUGR has been linked to the development of type 2 diabetes. Recent data suggest that some of the molecular defects underlying type 2 diabetes reside in the CNS. Disruption of the signal transducer and activator of transcription 3 (STAT3) in the hypothalamic neurons expressing leptin receptor, results in severe obesity, hyperglycaemia, and hyperinsulinemia. Our aim was to investigate the expression of STAT3 and its downstream effector proopiomelanocortin (POMC) in IUGR rats obtained by uterine artery ligation. On day 19 of gestation, time-dated Sprague-Dawley pregnant rats were anesthetized, and both the uterine arteries were ligated. At birth, hypothalamus was dissected and processed to evaluate the expression of STAT3, its phosphorylated form, and POMC. STAT3 mRNA, STAT3 protein, phosphorylated STAT3, POMC mRNA, and POMC protein were significantly reduced in IUGR versus sham animals (p < 0.0001, p < 0.05 and p < 0.001, p < 0.01, p < 0.01, respectively). No significant differences either in serum leptin concentrations or in hypothalamic leptin receptor expression were observed. Our results suggest that an abnormal intrauterine milieu can affect the hypothalamic expression of STAT3 and POMC at birth, altering the hypothalamic signaling pathways that regulate the energy homeostasis.

Central Control of Glucose Homeostasis

Type 2 diabetes is a complex disease mainly characterized by impaired insulin action and insulin secretion [1, 2]. A major breakthrough in the understanding of mechanisms leading to type 2 diabetes has recently come from studies on the central nervous system (CNS) dependent regulation of glucose and fat metabolism. The concept that the CNS has an essential role in glucose homeostasis and insulin action is not new. In 1849, Claude Bernard reported that pricking the floor of the fourth ventricle in rabbits produced hyperglycemia [3]. The past decade has greatly increased knowledge about the ability of the CNS to regulate food intake, body weight and glucose homeostasis. It is now clear that the CNS, and in particular the hypothalamus, plays a pivotal role in regulating glucose homeostasis independently of its effects on body weight. Therefore, some of the molecular defects underlying type 2 diabetes may reside in the CNS, supporting the concept that type 2 diabetes is, at least in part, a hypothalamic disorder.

Epigenetic Changes Predisposing to Type 2 Diabetes in Intrauterine Growth Retardation

Epidemiologic studies have demonstrated an association between intrauterine growth retardation and a greater risk of chronic disease, including coronary heart disease, hypertension, stroke, and type 2 diabetes in adulthood. An adverse intrauterine environment may affect both growth and development of the organism, permanently programming endocrine and metabolic functions. One of the mechanisms of programming is the epigenetic modification of gene promoters involved in the control of key metabolic pathways. The aim of this review is to provide an overview of the experimental evidence showing the effects of early exposure to suboptimal environment on epigenome. The knowledge of the epigenetic markers of programming may allow the identification of susceptible individuals and the design of targeted prevention strategies.

HSP70 production and inhibition of cell proliferation in Molt-4 T-cells after cell-to-cell transmission of HTLV-I: effect of PGA1.

Infection with HTLV-I is associated with leukemic transformation of mature CD4+ T lymphocytes. PGA1, a powerful inhibitor of tumour cell proliferation, can prevent the clonal expansion of HTLV-I-infected cells following acute infection of cord blood-derived mononuclear cells. Since the antiproliferative effect of PGA1 on HTLV-I transformed, chronically infected MT-2 cell line was associated with induction of HSP70, we have investigated the effect of PGA1 on cell cycle progression and HSP70 production in a leukemic T-cell line (Molt-4) shortly after exposure to HTLV-I in a cell-to-cell transmission model. Rate of cell proliferation and HSP70 expression were studied within one duplication cycle of Molt-4 cells after exposure to HTLV-I. Growth of both control and virus-exposed cultures was inhibited by treatment with PGA1 (4 micrograms/ml) and cell cycling was arrested preferentially at the G1/S interphase. Synthesis of HSP70 was induced within 3 h by PGA1 in control and virus-exposed Molt-4 cells and became undetectable from overnight onward, though the protein accumulated in the cells. The arrest of growth was observed from overnight up to 48 h so that treated cells almost missed one cycle. Interestingly, HSP70 transcript and protein persisted at remarkably high levels in Molt-4 cells exposed to HTLV-I in the absence of PGA1, showing that HSP70 expression can be directly activated during primary infection with this human retrovirus. Moreover, in these cocultures, treatment with PGA1 or heat shock was not able to increase further the elevated level of HSP70 found in untreated cocultures, suggesting that during the early period of the virus-transmission phase, HTLV-I could interfere with HSP70 induction by other inducers.

Serum Levels of Polybrominated Diphenyl Ethers in Girls with Premature Thelarche

Background/Aims: Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and have shown endocrine disruption properties in experimental studies. The aim of this study was to investigate the association between the exposure to PBDEs and alterations of puberty in girls referred for idiopathic central precocious puberty (ICPP) and premature thelarche (PT). Methods: A case-control study was conducted in 124 girls: 37 girls with ICPP (mean age 7.4 ± 0.9 years), 56 with PT (mean age 5.7 ± 2.1 years) and 31 controls (mean age 5.4 ± 1.9 years). PBDE serum concentrations, hormone levels and anthropometry were assessed. PBDE concentrations were corrected for total serum lipid content. Individual exposure to PBDEs was evaluated through ad hoc questionnaires. Results: PBDE serum concentrations corrected for total lipid content were significantly higher in girls with PT (mean 1.49 ± 0.63 log ng/g) than in controls (mean 1.23 ± 0.54 log ng/g; p < 0.05). PT girls showed higher levels of PBDE than ICPP girls (1.49 ± 0.63 vs. 1.37 ± 0.49 log ng/g), though this was not significant. An analysis of the questionnaires revealed no significant differences in exposure between the three groups. Conclusion: Our findings suggest that higher concentrations of serum PBDEs are associated with PT in girls.