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R Lauro

Publications and source records attributed to R Lauro.

At least 37 records · Page 2Linked to original sources
PubMed1996

Quantitative analysis of pancreatic glucokinase gene expression in cultured beta cells by competitive polymerase chain reaction.

Regulation of glucokinase (GK) gene expression in pancreatic beta cells has been poorly investigated, both due to low abundance of the gene and to difficulties in cells isolation. The present study describes the establishment of a competitive RT-PCR method for quantitative analysis of GK gene. The method has been applied to the analysis of GK mRNA expression RIN 1046-38 cells. We have monitored modifications of GK mRNA expression after different periods of time in culture and we have studied the effect induced by dexamethasone (DEX) treatment. We show that the method is very sensitive and requires very low amount of RNA. Data demonstrate that GK mRNA expression in RIN cells is reduced as a function of passages in culture and that the reduction is positively correlated with the decrease of insulin responsiveness observed in high passages cells. DEX treatment inhibits GK mRNA expression in RIN cells in a dose-dependent and time-dependent manner.

Animals↗
PubMed1996

Differential regulation of adrenocorticoid receptors in the hippocampus and spinal cord of adrenalectomized rats.

Using multiple polymerase chain reaction assay and cytosolic receptor binding assay we studied type I, mineralocorticoid receptor (MR), and type II, glucocorticoid receptor (GR), adrenocorticoid receptors expression in rat hippocampus and spinal cord, at various times after adrenalectomy: 12 hr, 24 hr, 3 days, and 1 week. Analysis of the data demonstrates that in hippocampus the expression of MR and GR mRNA was not significantly affected by adrenalectomy. On the contrary, Bmax of MR was significantly increased at each time post-surgery, with only slight modifications of Kd. Bmax and Kd for GR showed a significant increase after 3 days and 1 week. In the spinal cord, MR mRNA was increased 12 hr after adrenalectomy, reaching a maximum at 3 days. Bmax of MR was also significantly increased after 3 days, whereas its Kd remained unchanged for the entire duration of the the study. Both GR mRNA and binding parameters were poorly affected by adrenalectomy. The results of the present experiments demonstrate that the absence of adrenocortical hormones influences differentially MR and GR expression in hippocampus and spinal cord, suggesting the existence of various and independent mechanisms of regulation of adrenocorticoid receptor.

Adrenal Cortex Hormones↗
PubMed1996

Delayed intracellular dissociation of the insulin-receptor complex impairs receptor recycling and insulin processing in cultured Epstein-Barr virus-transformed lymphocytes from insulin-resistant subjects.

Insulin-receptor internalization and processing are defective in insulin-resistant subjects. To assess the reversibility of these defects, we cultured Epstein-Barr virus-transformed-lymphoblasts from six normal, six obese, and six non-insulin-dependent diabetic (NIDDM) subjects in media containing low (5 mmol/l) or high (25 mmol/l) glucose concentrations, and studied the insulin-receptor internalization and processing in vitro. In cells from normal, obese, and NIDDM subjects cultured in low glucose concentrations, exposure to 100 nmol/l insulin for 30 min at 37 degrees C reduced cell-surface 125I-insulin binding to a similar extent (82 +/- 2, 77 +/- 5, and 82 +/- 5% of initial values, respectively). The same results were obtained with cells cultured in high glucose concentrations. In cells cultured under both glucose conditions, and exposed to 100 nmol/l insulin for 30 min at 37 degrees C, a complete recovery of the initial 125I-insulin binding was observed in normal but not in obese and NIDDM subjects. Release of intracellular insulin and its degradation in vitro was determined by incubating cells with 600 pmol/l of 125I-insulin for 60 min at 37 degrees C, acid washing cells, and re-incubating in insulin-free buffer at 37 degrees C. The radioactivity released by cells was characterized by trichloroacetic acid precipitability, Sephadex G-50 column chromatography, and re-binding to fresh cells. Rates of release of internalized radioactivity were reduced in obese and NIDDM subjects (t1/2 = 61 +/- 9 min, p < 0.02; 58 +/- 10 min, p < 0.05; and 38 +/- 4 min in obese, NIDDM, and normal subjects, respectively). The percentage of intact insulin released from cells was significantly higher in obese and NIDDM subjects than in the normal subjects. The t1/2 of intracellular dissociation of insulin-receptor complexes measured by a polyethylene glycol assay was lower in normal (6 +/- 1 min) than in obese (12 +/- 2 min, p < 0.03) and NIDDM subjects (14 +/- 3 min, p < 0.02). The results suggest that in insulin-resistant subjects a primary defect in intracellular dissociation of insulin is responsible for alterations of receptor recycling and insulin processing.

Adult↗
PubMed1996

Effect of biotin on glucokinase activity, mRNA expression and insulin release in cultured beta-cells.

Biotin is known to influence hepatic glucokinase (GK) expression both at a transcriptional and at a translational level. The aim of the present paper was to investigate the effect of biotin on pancreatic GK. For this purpose, RIN1046-38 cells were cultured in the presence of different biotin concentrations for different times; there-after, GK mRNA expression, GK activity and insulin release were studied. Results demonstrated that biotin has a biphasic effect on GK mRNA expression, being stimulatory after short-term treatment and inhibitory after longterm treatment. GK activity was increased after long-term treatment. Insulin release was not affected by biotin treatment. These data suggest that biotin may influence glucose metabolism also by acting directly at the level of beta-cells.

Animals↗
PubMed1996

Atrial natriuretic factor (ANF) and ANF receptor C gene expression and localization in the respiratory system: effects induced by hypoxia and hemodynamic overload.

Atrial natriuretic factor (ANF) and ANF receptor C (ANF.RC) expression have been investigated in healthy and cardiomyopathic hamsters (CMPH) with widespread necrosis of the diaphragm and myocardium leading to respiratory and heart failure. ANF- and ANF.RC-producing cells were localized in different structures of the respiratory system, and the regulation of their expression by the individual and/or combined action of hypoxia and hemodynamic overload was analyzed. The study was performed in 20-, 90-, and 150-day-old animals using immunohistochemistry, in situ hybridization, Northern blot, and RIA analyses. ANF was shown to be expressed in the tracheo-bronchial epithelium and muscle and, to a lesser extent, in the alveolar wall and muscular media of the pulmonary arteries and extraparenchymal pulmonary veins in both healthy hamsters and CMPH. In 150-day-old CMPH, hypoxia (PaO2 < 50 mm Hg) induced a 10-fold increase in ANF messenger RNA accumulation and a 6-fold increase in the immunoreactive ANF (IR-ANF) concentration in lungs, as quantitated by RIA. As plasma IR-ANF concentrations were elevated in all CMPH age groups, it was most likely produced by the myocardium. ANF.RC messenger RNA was homogeneously distributed throughout the entire respiratory system and was increased 2-fold in hypoxic 150-day-old CMPH only. These results suggest that ANF originating in the respiratory system exerts only paracrine effects on different structures of the respiratory system in addition to the action of circulating ANF. Hemodynamic overload (left ventricular end-diastolic pressure, 17.20 +/- 3.80 mm Hg) might contribute to enhanced ANF gene expression only in extraparenchymal pulmonary vein walls of 150-day-old CMPH. We also propose that ANF.RC overexpression might be a protective mechanism operated via either ANF clearance or inhibition of adenylate cyclase activity to counteract exaggerated smooth muscle relaxation.

Animals↗
PubMed1995

Distribution of glucocorticoid receptor mRNA in the rat spinal cord.

Using in situ hybridization (ISH), we studied the distribution of rat glucocorticoid receptors (GR) mRNA in rat spinal cord. mRNA encoding for GR was abundant throughout the white matter and a clear pattern of distribution was detected within the grey matter. In the grey matter mRNA was primarily localized in the ventral horn, where motoneurones were strongly labelled. In the dorsal horn, the distribution appears more diffuse but the superficial layers (I and II) clearly exhibited a shigher signal. We conclude that, in rat spinal cord, GR are present in both glial and neuronal cells. In particular, both somatosensory and motor pathways contain GR.

Animals↗
PubMed1995

Peptide-based radioimmunoassay for the two isoforms of the human insulin receptor.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. In this study, we developed a radioimmunoassay for the two isoforms employing antibodies raised against two peptides, one (Pep-12) corresponding to residues encoded by exon 11, and the other (Pep-13) corresponding to a COOH-terminal domain of the alpha-subunit which is common to both HIR-A and HIR-B isoforms. These peptides were iodinated and used as both ligands and standards. The assay is specific, highly reproducible, and sensitive with a detection limit of 10 fmol of receptor. One mole of purified insulin receptor, measured by Scatchard analysis, is read as one mole of receptor in the radioimmunoassay with either Pep-12 or Pep-13 as standards. The radioimmunoassay is applicable to the measurement of total content and relative abundance of the two isoforms in extracts from various tissues. We applied the radioimmunoassay to measure the relative abundance of the two isoforms in fat and muscle from normal, obese non-diabetic and non-insulin-dependent diabetic (NIDDM) subjects. Results demonstrate that expression of the low-affinity HIR-B form is significantly increased in obese and NIDDM subjects compared with control subjects. In addition, the increased expression of the HIR-B isoform was significantly correlated with both body mass index (r = 0.52; p = 0.006) and fasting glucose levels (r = 0.59; p = 0.001).

3T3 Cells↗
PubMed1995

Efficacy of low-dose GnRH analogue (Buserelin) in the treatment of hirsutism.

The aim of the present study was to evaluate the effect of low dose GnRH analogue (Buserelin) on gonadal steroid secretion and hair growth in hirsute women. The drug was administered as a nasal spray (200 micrograms tid) to reduce gonadal steroid secretion. Eight hirsute women were treated for six month with the gonadotropin-releasing hormone analog. All had subclinical polycystic ovary syndromes on the basis of ultrasound or hormonal data, together with ovary dysfunctions and irregular menses. None had adrenal or pituitary dysfunction. The score of hirsutism was evaluated according to Ferriman and Gallway; pituitary function was evaluated measuring the FSH and LH response to GnRH stimulation and gonadal steroid secretion by measuring estradiol, progesterone, total plasma testosterone, androstenedione and androstanediol. Sex hormone binding globulin, insulin, prolactin and DHEA-S were also measured. The suppression of ovarian steroid secretion was confirmed by reductions in total plasma testosterone, and rostenedione and androstanediol that were detectable after one month of treatment. FSH and LH responses to GnRH stimulation were inhibited consistent with pituitary desensitization. No significant side effects were observed and all patients completed the trial. The score of hirsutism was 24 +/- 5 before, 19.6 +/- 6 by the 3rd month and 16.8 +/- 5.1 by the 6th month of treatment (p < 0.001); the effect was still evident 1 and 6 months after the withdrawal of the therapy (14.8 +/- and 15.8 +/- 5 respectively; p < 0.001). Our findings indicate that Buserelin is useful in the treatment of non adrenal hirsutism when other forms of therapy are contraindicated or poorly tolerated by the patient.

Adolescent↗
PubMed1994

Molecular and cellular characterization of the GABAA receptor in the rat pancreas.

In the present study, we characterize the molecular structure of the GABAA receptor in pancreas, islets, alpha and beta cells, and in RIN 1046-38 cells. Using the polymerase chain reaction and specific primers for 11 out of the 15 subunits known so far, that may contribute to the composition of the GABAA receptors, we demonstrate that pancreas and its cellular components, as well RIN 1046-38 cells, might contain a GABAA receptor resulting from all the possible combinations in a pentameric configuration of the subtypes alpha 1, alpha 2, alpha 3 of the alpha subunit family, beta 1, beta 2, beta 3 subtypes of the beta subunit family, delta subunit and gamma 2 subtype of the gamma subunit family. The presence of the gamma 2 subunit renders the GABAA receptors potentially sensitive to allosteric modulators.

Animals↗
PubMed1994

Role of the exon 11 of the insulin receptor gene on insulin binding identified by anti-peptide antibodies.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the C-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. It was shown that the two isoforms exhibit different binding affinities for insulin, thus suggesting that the sequence encoded by exon 11 may be important for insulin binding. To further investigate this issue, we generated polyclonal antibodies against C-terminal peptides of the two HIR alpha-subunit variants. Herein, we characterized two antibodies, PA-11 and PA-12, directed against the C-terminus or the N-terminus of the sequence encoded by exon 11, respectively, and one (PA-13) directed against a sequence in the carboxy-terminal region of the alpha-subunit which is common to HIR-A and HIR-B. Antibodies were characterized for their ability to immunoprecipitate the receptor and to inhibit [125I]insulin binding to both isoforms. We found that PA-13 immunoprecipitates both the HIR-A and the HIR-B, PA-12 immunoprecipitates exclusively the HIR-B, and PA-11 fails to precipitate both isoforms. Interestingly, PA-12 inhibits specifically insulin to the HIR-B, whereas other PAs fail to affect insulin binding to either isoforms. Furthermore, PA-12 linearises the Scatchard plot of binding data, and retards the dissociation rate of insulin, thus suggesting that antibody affects cooperative interactions among binding sites. We conclude that the sequence encoded by exon 11 may play a role in modulating the binding of insulin to the receptor and negative cooperativity.

3T3 Cells↗
PubMed1994

Variant sequences of insulin receptor substrate-1 in patients with noninsulin-dependent diabetes mellitus.

The pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM) is characterized by insulin resistance and insulin deficiency. To search for genetic defects causing NIDDM, we have screened for mutations in the gene encoding insulin receptor substrate-1 (IRS-1), an intracellular protein that is phosphorylated by the insulin receptor and is thought to play an important role in mediating insulin action. The coding sequence of the IRS-1 gene (divided into 12 overlapping fragments) was amplified by polymerase chain reaction and screened for the presence of single stranded conformational polymorphisms. This led to the identification of 6 variants in the nucleotide sequence. There were 3 nonconservative amino acids substitutions: Gly819-->Arg, Gly972-->Arg, and Arg1221-->Cys. In addition, there were three silent polymorphisms: GAC vs. GAT encoding Asp90, GGG vs. GGA encoding Gly235, and GCA vs. GCG encoding Ala805. The previously reported Arg972 substitution was identified in 7 of 31 patients with NIDDM, 4 of 32 normal subjects, and 4 of 16 nondiabetic obese individuals. The 2 novel amino acid substitutions (Arg819 and Cys1221) were both detected in 1 patient with NIDDM, but not in either of the other 2 groups of nondiabetic individuals. All 3 amino acid residues are identically conserved in the amino acid sequences of human, mouse, and rat IRS-1, suggesting that Gly819, Gly972, and Arg1221 are important for the normal function of IRS-1. Furthermore, the prevalence of amino acid substitutions in IRS-1 is increased in patients with NIDDM. These observations suggest that mutations in the IRS-1 gene may play a causal role in the pathogenesis of NIDDM.

Adult↗
PubMed1992

Androgens increase insulin receptor mRNA levels, insulin binding, and insulin responsiveness in HEp-2 larynx carcinoma cells.

Androgen receptors have been found in human larynx and androgens have been supposed to play an important role in promoting the growth of laryngeal carcinomas. The molecular mechanism underlaying this phenomenon is not at all understood. Aim of this work was to investigate the effects of two androgens (testosterone and dihydrotestosterone) on insulin receptor mRNA levels and insulin binding activity as well as on either metabolic or growth-promoting actions of insulin in a human larynx carcinoma cell line (HEp-2). We found that HEp-2 cells express a high affinity insulin receptor. Both androgens significantly increase insulin receptor mRNA levels and insulin receptor number in HEp-2 cells. Insulin action, evaluated either as total glucose utilization or as [3H]thymidine incorporation into DNA, significantly increased in HEp-2 treated with androgens in comparison to control cultures. Altogether, our data allow us to speculate that the increased insulin effectiveness we observed in the larynx carcinoma cell line HEp-2 after androgen treatment might be involved in the regulation of larynx cancer cells growth.

Carcinoma, Squamous Cell↗
PubMed1992

Evidence that two naturally occurring human insulin receptor alpha-subunit variants are immunologically distinct.

The IgG from a patient (Italy 2 [I2]) with hypoglycemia, due to autoantibodies to the insulin receptor, was purified on protein A Sepharose into two fractions that were tested in various human tissues and cells. The IgG fraction that bound protein A (absorbed IgG [IgGa]) nearly completely inhibited the binding of 125I-labeled insulin to various cells or tissues (placenta, IM-9, adipocytes, HEp-2-larynx cells, Epstein-Barr virus lymphocytes) but not greater than 50% of 125I-labeled insulin binding to human liver membranes. Conversely, both the IgG fraction from this patient, which did not bind protein A (flow-through IgG [IgGb]), and the IgGa fraction from a second similar patient (Italy 1 [I-1]) almost completely inhibited the binding of 125I-labeled insulin to liver membranes. The IgGa fraction from patient I-2 did not change receptor affinity because 50% inhibition of 125I-labeled insulin binding was not affected by either the presence or absence of these IgG fractions. Furthermore, liver binding data were not due to cross-reaction of 125I-labeled insulin to the insulinlike growth factor I receptor, and treatment of liver membranes with neuraminidase did not alter the inhibitory effect of the IgGa fraction from patient I-2 on 125I-labeled insulin binding to liver. Binding inhibition experiments performed with cells transfected with and overexpressing the -12 (human insulin receptor [HIR]-A) or the +12 (HIR-B) variant of HIR revealed that the IgGa fraction from patient I-2 inhibited 125I-labeled insulin binding to the HIR-A receptor but not to the HIR-B receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1991

Altered expression of the two naturally occurring human insulin receptor variants in isolated adipocytes of non-insulin-dependent diabetes mellitus patients.

The human insulin receptor gene is expressed in two variant isoforms which differ by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the extracellular alpha-subunit as a consequence of alternative splicing of exon 11. Expression of the two variant isoforms is regulated in a tissue-specific manner. In this study, we have measured the levels of the two receptor variants in isolated adipocytes from 10 non-insulin-dependent diabetes mellitus (NIDDM) and 11 normal subjects using an immunological assay, based on the ability of a human anti-receptor autoantibody to discriminate between HIR-A and HIR-B. Results indicate that levels of HIR-B variant are increased in NIDDM patients.

3T3 Cells↗
PubMed1991

Evidence that human and porcine insulin differently affect the human insulin receptor: studies with monoclonal anti-insulin receptor antibodies.

Binding studies have been carried out with radioiodinated monoclonal antibodies directed to various epitopes of the insulin receptor in order to detect differences between human and porcine insulin in the interaction with the human insulin receptor. Human insulin was more effective that porcine insulin at inhibiting the binding of 125I-MA-5 to IM-9 cells, Hep-2 human larynx cells and human placenta membranes. On the contrary, human and porcine insulin showed similar inhibitory effect on the binding of two other labeled anti-insulin receptor monoclonal antibodies, thus ruling out the possibility that results were due to experimental artifacts. Although several interpretations are possible, data reported suggest that human insulin and porcine insulin might differently affect the insulin receptor, even if, the biological significance of these findings remains unknown.

Animals↗
PubMed1991

Modulation of insulin secretion by diazepam binding inhibitor and its processing products.

The effect of purified rat brain diazepam binding inhibitor (DBI) and that of synthetic DBI fragments DBI33-50 [octadecaneuropeptide (ODN)], DBI17-50 [triakontatetraneuropeptide (TTN)], DBI42-50 and DBI53-62 were studied on glucose-induced secretion of insulin from isolated rat pancreatic islets in both static incubation and perifusion experiments. DBI and ODN did not affect the secretion of insulin in the presence of 2.8 mM glucose (basal condition) but reduced the release of insulin induced by 16.7 mM glucose. The effects of DBI and ODN were significant at concentrations as small as 1-10 nM. In contrast, 100 nM TTN, DBI42-50 (which corresponds to the COOH-terminal region of ODN) and DBI53-62 (which corresponds to a region of DBI that is conserved among species), were without effect on both basal and 16.7 mM glucose-stimulated insulin secretion. DBI has previously been localized to the delta cells of islets of Langerhans (Ostenson, Ahren, Karlsson, Sandberg and Efendic, 1990) and the presence of DBI- and ODN-like immunoreactivity in isolated rat pancreatic islets has now been demonstrated. These results suggest that DBI and some of its processing products (ODN) may modulate glucose-stimulated secretion of insulin through a paracrine mechanism.

Animals↗