Luisella Alberti

Expression of long pentraxin PTX3 in human adipose tissue and its relation with cardiovascular risk factors

UNLABELLED Pentraxin 3 (PTX3) is an acute phase protein strongly expressed by advanced atherosclerotic lesions. We investigated (a) PTX3 expression and secretion in subcutaneous adipose tissue (SAT) and omental visceral adipose tissue (VAT) obtained from 21 obese (37.4+/-8.15 yr) and 10 normal weight subjects (43.7+/-11.07 yr) and (b) the relationships of adipose PTX3 with tumour necrosis factor alpha (TNFalpha) and adiponectin expression and with cardiometabolic risk factors. Real-time PCR was used to quantify specific mRNA for PTX3, CD68 (macrophage marker), TNFalpha and adiponectin. Fresh adipose tissue was cultured and PTX3 measured in the medium. Serum insulin, glucose, HDL and LDL cholesterol, triglycerides, C-reactive protein (CRP), fibrinogen, adiponectin, TNFalpha and PTX3 were measured. PTX3 expression was similar in the two fat compartments and tended to be higher in obese than in normal weight subjects in VAT only (p=0.05). CD68 and PTX3 expressions were correlated with each other in SAT but not in VAT. After adjustment for age and sex, VAT-PTX3 expression and release were correlated with VAT-TNFalpha expression (p<0.01 for both) and with LDL/HDL ratio (p<0.01 and p<0.001). VAT-PTX3 expression was also correlated with BMI, triglycerides, CRP, fibrinogen and adiponectin (p<0.05 for all). In the multivariate analysis with VAT-PTX3 RNA levels as dependent variable, LDL/HDL ratio and fibrinogen remained independently associated with VAT-PTX3 expression (p<0.01 for both). These associations were not seen within SAT. CONCLUSIONS Human adipose tissue expresses and releases PTX3 likely under TNFalpha control. VAT production of PTX3 seems to contribute to the mechanisms underlying the development of atherosclerosis.

Multigenerational familial medullary thyroid cancer (FMTC): evidence for FMTC phenocopies and association with papillary thyroid cancer

background Occurrence in a familial setting is well established for medullary thyroid carcinoma (MTC) and has been more recently reported for papillary thyroid cancer (PTC). Germline mutations or rearrangements of the RET proto‐oncogene are the genetic background of the majority of hereditary MTCs and of about 25–40% of PTCs.

RET genotypes in sporadic medullary thyroid cancer : studies in a large Italian series

Background  Highly discrepant data about the different distribution of RET germline single nucleotide polymorphisms (SNPs) among patients with sporadic medullary thyroid cancer (sMTC) and controls are available.

Protein Kinase A Regulatory Subunits in Human Adipose Tissue Decreased R2B Expression and Activity in Adipocytes From Obese Subjects

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects. RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively. RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects. CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity.

A novel missense mutation in the signal peptide of the human POMC gene: a possible additional link between early-onset type 2 diabetes and obesity.

Rare mutations in several genes have a critical role in the control of homeostatic mechanisms such as food-intake, energy balance and glucose metabolism. In this study, we performed a mutational screening in a 58-year-old woman presenting early-onset type 2 diabetes and central obesity. The entire coding regions of MC4R, MC3R, HNF1A, GCK and POMC (pro-opiomelanocortin) genes were analyzed by direct sequencing. A new missense mutation was identified within the POMC gene signal peptide sequence, resulting in a heterozygous substitution of an arginine for a glycine at codon 15 (p.A15G) that was excluded in 300 healthy normal weight controls. The mutation segregated in the family and was associated with overweight, type 2 diabetes, hypertension and coronary heart disease in the carriers. Functional studies demonstrated that POMC protein was not detectable in β-TC3 cells transfected with A15G-POMC vector as well as in their culture media, despite POMC mRNA levels were comparable for amount and stability to those of wild-type-transfected cells. In silico RNA folding prediction indicated that the mutation gives rise to a different RNA secondary structure, suggesting that it might affect translation and protein synthesis. To the best of our knowledge, this is the first report addressing the functional consequences of a mutation in the signal peptide of POMC. These findings further support the hypothesis that POMC-derived peptides might have a role in the control of peripheral glucose metabolism and suggest that disruption of central POMC secretion might represent an additional link between type 2 diabetes and obesity.

In vitro and in vivo effects of metformin on human adipose tissue adiponectin.

Objective: The effects of metformin on adiponectin production are controversial and have never been investigated in human adipose tissue. We analysed whether metformin modulates, in vitro and in vivo, gene expression, protein content, and secretion of adiponectin. Methods: For the in vitro study, subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) samples from 5 non-diabetic obese patients were collected. For the in vivo investigation, 22 obese patients were randomly assigned to metformin+lifestyle (ML) or placebo+lifestyle (PL) intervention. SAT specimens and blood samples were collected before and after the intervention in both groups. Results: In in vitro experiments, treatment with metformin increased the expression and secretion of adiponectin in SAT, but not in VAT explants. In the in vivo study, a significant increase in adiponectin and a decreased expression of a macrophage activation marker (CD68) were observed only in SAT of the ML group. Conclusion: These results demonstrate that metformin is able to up-regulate adiponectin gene expression, both in vivo and in vitro, and to stimulate adiponectin protein secretion from human SAT in vitro. It could be hypothesised that metformin-induced adiponectin increase within adipose tissue may have an unexpected role in the reduction of local inflammation.

HESX1 expression in human normal pituitaries and pituitary adenomas

Hesx1 is a paired-like homeobox gene first expressed during mouse embryogenesis in the anterior midline visceral endoderm. As gastrulation proceeds, Hesx1 is expressed in the ventral prosencephalon and, subsequently, at E9.0 appears in the ventral diencephalon and in the thickened layer of oral ectoderm that give rise to Rathkes pouch, the primordium of the anterior pituitary gland. Hesx1 continues to be expressed in the developing anterior pituitary until E11.5 when its transcripts disappear in a spatiotemporal sequence corresponding to progressive pituitary cell differentiation, becoming undetectable by E15.5. In the present study, we investigated whether HESX1 is expressed during adult life in human normal pituitaries and in different types of human pituitary adenomas. We analysed, using quantitative RT-PCR method, three normal pituitaries, seven GH-, two TSH-, two PRL-, one ACTH-secreting adenomas, and seven nonfunctioning pituitary tumors. HESX1 mRNA was found to be expressed in normal pituitaries and in all the pituitary tumors that we have analysed. These results suggest that in humans HESX1 is not turned-off during the adult life as it occurs in mice. Thus, HESX1 in humans might play a role in the maintenance of the anterior pituitary cell types and function, as well as in the differentiation of pituitary adenomas, whose pathogenetic mechanisms remain to be further investigated. This is the first study on HESX1 expression in humans during adult life.

Absence of thyroid transcription factor-1 expression in human parathyroid and pituitary glands.

Thyroid transcription factor-1 (TTF-1), a tissue-specific nuclear transcription factor involved in the embryogenesis and differentiation of human thyroid, lung and brain, has been recently identified in other rat tissues, including parafollicular C cells and parathyroid chief cells. Based on this distribution, a possible role for this factor in calcium homeostasis has been suggested. This study investigated the presence of TTF-1 transcripts and protein in human tissues expressing the calcium sensing receptor (CaSR). Using a RT-PCR technique, complemented by Southern blot analysis, TTF-1 expression was detected in human C cells (two medullary thyroid carcinomas), but not in normal and adenomatous (four adenomas and three hyperplasia) parathyroid, and normal and adenomatous (six adenomas) pituitary tissues. CaSR was expressed in all samples. The absence of expression was confirmed by Western blot. In contrast to previous studies in the rat, this study demonstrates the absence of TTF-1 transcripts in the human adult parathyroid and pituitary glands, although a role for this factor during the ontogeny of these organs cannot be excluded.