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Biomedical subjects

A Brunetti

Publications and source records attributed to A Brunetti.

At least 73 records · Page 4Linked to original sources
PubMed1991

Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163.

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.

Aminoisobutyric Acids↗
PubMed1991

Relative protection from cerebral atherosclerosis of young patients with homozygous familial hypercholesterolemia.

It is well known that hypercholesterolemia is correlated with coronary atherosclerosis, but no definite information is available on its association with cerebrovascular atherosclerosis. We studied 10 young patients (age 3-32 years) with homozygous familial hypercholesterolemia (FH), together with 3 normal relatives as healthy controls. Extra- and intracranial Doppler examination, MRI and cerebral blood flow by SPECT and 99mTc-HM-PAO were performed on all. Six out of 10 patients already had signs and symptoms of coronary heart disease, but all patients were free from ischemic brain lesions, as small as detectable at MRI, and had normal cerebral blood flow. Two patients presented significant stenosis of the carotid arteries at Doppler examination. Young patients with homozygous FH have early and clinically evident coronary atherosclerosis, while overt disease in the cerebral district is delayed despite the extremely elevated plasma cholesterol concentration. This was also confirmed by the autopsy of two patients, who died after the study and whose cerebral arteries were totally free from atherosclerotic lesions. The age, at which flow-reducing atherosclerotic lesions develop in hypercholesterolemic patients, differs with regard to the arterial district involved.

Adolescent↗
PubMed1991

Regulation of insulin-like growth factor (IGF) I receptor expression during muscle cell differentiation. Potential autocrine role of IGF-II.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. The presence of specific, high affinity IGF receptors, as well as the expression of IGF peptides and binding proteins by muscle suggest that a significant component of IGF action in this tissue is mediated through autocrine and/or paracrine mechanisms. To explore autocrine/paracrine action of IGFs in muscle, we studied the regulation of the IGF-I receptor and the expression of IGF peptides during differentiation of the mouse BC3H-1 muscle cell line. Differentiation from myoblasts to myocytes was associated with a 60% decrease in IGF-I receptor sites determined by Scatchard analysis. Analysis of mRNA abundance and protein labeling studies indicated that the decrease in IGF-I receptor sites was associated with similar reductions in IGF-I receptor gene expression and receptor biosynthesis. IGF-II peptide gene expression was detected in myoblasts and increased 15-fold with differentiation; the increase in IGF-II gene expression preceded the decrease in IGF-I receptor gene expression. In contrast, IGF-I peptide gene expression was low in myoblasts and decreased slightly with differentiation. To explore the potential role of endogenous IGF-II in the differentiation-associated decrease in IGF-I receptor expression, we investigated the effects of IGF-II treatment in myoblasts. The addition of IGF-II to undifferentiated myoblasts resulted in downregulation of the IGF-I receptor which was associated with decreased IGF-I receptor biosynthesis and decreased IGF-I receptor mRNA abundance. These studies suggest, therefore, that IGF-I receptor expression during muscle cell differentiation may be regulated, at least in part, through autocrine production of IGF-II.

Animals↗
PubMed1991

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.

Animals↗
PubMed1991

[131I-meta-iodobenzylguanidine scintigraphy in patients with a suspected pheochromocytoma. A comparison with CT and biohumoral parameters].

The results of 131I metaiodobenzylguanidine (MIBG) and Computed Tomography (CT) scans in a group of patients with clinically suspected pheochromocytoma were evaluated and compared with biohumoral parameters. We studied 24 consecutive patients (7 M and 17 F; age range 20-66 years). 131I-MIBG scintigraphy and CT were in agreement in 19 patients (79%): of them 7 cases were true positive, and 12 were true negative. Disagreement between the two imaging techniques was observed in 5 patients (21%). In this group, one patient, with positive CT scan, had false negative MIBG study, while 4 patients with negative MIBG scan, had false positive CT. MIBG showed significantly higher (p less than 0.05) specificity (100%), positive predictive value (100%), and accuracy (96%) than CT (75%, 67%, and 83%, respectively). 131I-MIBG scintigraphy is an accurate, noninvasive technique for localizing pheochromocytoma and providing direct tissue characterization, while CT provides more accurate spatial information. In conclusion, CT and MIBG studies are complementary in the evaluation of patients with suspected pheochromocytoma.

3-Iodobenzylguanidine↗
PubMed1990

Progestins increase insulin receptor content and insulin stimulation of growth in human breast carcinoma cells.

The effects of progesterone on the growth of breast carcinoma cells are undefined. In the present study we investigated the effect of progestins on insulin receptor gene expression and insulin action in human breast cancer cells. Treatment of T47D cells with the synthetic progestin R5020 induced a time- and dose-dependent increase in insulin receptor content as measured by both ligand-binding studies and radioimmunoassay. Binding was half-maximally stimulated at 300 pM R5020 and maximal levels were reached after 4 days of treatment. Progesterone was 10-fold less potent than R5020. Cortisol had no effect on insulin receptor levels, while 17 beta-estradiol and dihydrotestosterone had minimal effects. Progestin treatment both increased insulin receptor mRNA levels and altered the relative distribution of the multiple insulin receptor mRNA transcripts. In order to study the functional significance of the increased insulin receptor levels, we incubated T47D cells with progesterone and then treated them with insulin. Insulin alone had a small effect on cell growth; however, the effect of insulin was markedly potentiated by progesterone treatment. These studies in breast cancer cells demonstrate, therefore, that insulin receptor gene expression is under the regulation of progestins and raise the possibility that progestin-insulin interactions may regulate breast cancer cell growth in vivo.

Antibodies, Monoclonal↗
PubMed1990

Role of myogenin in myoblast differentiation and its regulation by fibroblast growth factor.

In BC3H-1 cells, a model for studying the events that occur when myoblasts transform into myocytes, we observed that differentiation was associated with a 10-20-fold increase in both gene transcription and mRNA levels for myogenin, a member of the myc gene family. In contrast, the expression of the related protein MyoD1 was not influenced. This enhanced expression of myogenin was followed by morphological differentiation and increased expression of the nicotinic acetylcholine receptor, a muscle-specific gene product. Exposure of BC3H-1 cells to a 20-base myogenin antisense oligomer blocked morphological differentiation and resulted in nearly complete inhibition of acetylcholine receptor protein expression. To further study the relationship between muscle cell differentiation and myogenin gene expression, fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation, was employed. FGF treatment inhibited myogenin gene transcription and BC3H-1 cell differentiation. These results demonstrate therefore that myogenin is an important regulator of skeletal muscle cell differentiation, and the expression of myogenin is under the control of FGF.

Animals↗
PubMed1990

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor.

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.

Animals↗
PubMed1990

Pituitary microadenomas: a PET study.

Twenty cases of surgically verified pituitary microadenoma (17 with Cushing disease and three with acromegaly) were studied with positron emission tomography (PET) with use of fluorine-18-2-fluorodeoxyglucose (FDG). The diagnostic results were compared with those of other modalities, namely, computed tomography (CT), magnetic resonance (MR) imaging, and, in the cases of Cushing disease, simultaneous bilateral inferior petrosal sinus sampling (SIPS). The PET results showed 12 positive readings and one questionable reading, compared with seven positive readings and one questionable reading for CT (18 cases studied) and 13 positive and two questionable MR imaging readings. PET complemented MR imaging, in the sense that five of the positive PET readings were negative or questionable at MR imaging. PET studies of 20 healthy control subjects showed no false-positive cases, whereas other studies of healthy subjects with contrast material-enhanced CT and MR imaging have yielded, respectively, 20% and 15% positive readings, with findings suggestive of silent or occult adenomas.

Adenoma↗
PubMed1990

Role of iron and ferritin in MR imaging of the brain: a study in primates at different field strengths.

The authors measured in vivo signal intensity on magnetic resonance (MR) images and postmortem iron concentrations in the brains of three young and two old rhesus monkeys. T2-weighted MR imaging was done at 0.5, 1.5, 2.0, and 4.7 T. Relative assessment of iron concentration was made from the optical density of brain sections stained with the Perls' method intensified with diaminobenzidine. MR imaging and optical density measurements were made in the centrum semiovale (white matter) and in four gray matter areas: the insular cortex, caudate nucleus, putamen, and globus pallidus, the latter three of which accumulate significant iron deposits with age. High optical density and decreased signal intensity were found in these areas, and the inverse correlation between gray matter/white matter signal ratio and optical density was in good agreement with the theory of T2 shortening caused by diffusion of water through magnetic inhomogeneities. However, the dependence of T2 shortening on field strength was not quadratic, as expected for paramagnetic iron, but instead showed a marked leveling off at higher field strengths. This magnetic "saturation" is explainable by antiferromagnetism and superparamagnetism of the ferritin core and has been observed in ferritin solutions at low temperatures. Similar observations at body temperature are needed before the iron-ferritin explanation for T2 shortening can be considered proved.

Aging↗
PubMed1990

Cerebral blood flow in experimental ischemia assessed by 19F magnetic resonance spectroscopy in cats.

We evaluated a 19F magnetic resonance spectroscopic technique that detects Freon-23 washout as a means of measuring cerebral blood flow in halothane-anesthetized adult cats during and after transient cerebral ischemia produced by vascular occlusion. The experiments were performed to test the ability of this recently developed method to detect postischemic flow deficits. Results were consistent with postischemic hypoperfusion. The method also proved valuable for measuring small residual flow during vascular occlusion. Our experiments indicate that this method provides simple, rapid, and repeatable flow measurements that can augment magnetic resonance examinations of cerebral metabolic parameters in the study of ischemia.

Animals↗
PubMed1990

Differential effects of fibroblast growth factor on insulin receptor and muscle specific protein gene expression in BC3H-1 myocytes.

BC3H-1 mouse muscle cells in culture were employed to study the mechanisms which regulate insulin receptor gene expression during differentiation. When BC3H-1 myoblasts were plated in low serum media (1% fetal bovine serum), cell division ceased. Within 1 week cells had the morphological features of myocytes and expressed muscle specific proteins such as creatine phosphokinase and the nicotinic acetylcholine receptor. It is known that following incubation in low serum media, the steady state mRNA levels for the key muscle transcription factor, myogenin are increased. Exposure of BC3H-1 cells to a 20-base myogenin antisense oligomer blocked morphological differentiation, and resulted in nearly complete inhibition of the expression of the acetylcholine receptor but not the insulin receptor(IR). In order to study further the relationship between differentiation and IR gene expression, fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation, was employed. FGF treatment inhibited the transcription of both myogenin and the acetylcholine receptor. However FGF did not inhibit the transcription of the IR. These studies indicate therefore that IR transcription increases during muscle cell differentiation, and suggest that during differentiation the control of IR gene expression differs from the control of muscle specific proteins.

Animals↗
PubMed1990

Improvement with metformin in insulin internalization and processing in monocytes from NIDDM patients.

This study investigated the relative effect of obesity alone and in combination with non-insulin-dependent diabetes mellitus (NIDDM) on the intracellular processing of insulin and evaluated the effect of metformin therapy on this process. Monocytes from 11 obese hyperinsulinemic subjects, 13 obese hyperinsulinemic NIDDM patients, and 7 nondiabetic control subjects were incubated with A14-125I-labeled insulin for 60 min at 37 degrees C, and intracellular insulin degradation was characterized by high-performance liquid chromatography. Total cell-associated insulin (insulin binding) and internalized and degraded insulin were decreased in obese subjects and significantly decreased in obese NIDDM patients compared with nondiabetic control subjects. In NIDDM patients, intracellular insulin degradation was inversely correlated with fasting plasma glucose (P less than 0.01). Eight obese subjects and 9 obese NIDDM patients were restudied after 4 wk of therapy with metformin (850 mg twice a day). Plasma levels of the drug were superimposable in the two groups. Metformin therapy did not change glucose and insulin levels in obese subjects but caused a decrease in blood glucose in obese NIDDM patients. Total cell-associated radioactivity (insulin binding) significantly increased in both groups (P less than 0.01). On the contrary, internalized radioactivity increased (0.83 +/- 0.3 vs. 1.31 +/- 0.35%, P less than 0.01), and similarly, insulin degradation was enhanced (54.6 +/- 8.9 vs. 74.22 +/- 9.15%, P less than 0.01) only in monocytes from obese NIDDM patients. However, the levels of these parameters were still lower than in control subjects (internalization, 2.94 +/- 0.68%; degradation, 93.03 +/- 3.7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1990

Regulating insulin-receptor-gene expression by differentiation and hormones.

Insulin regulates cell function by first binding to the insulin receptor (IR) localized on the cell surface. With the cloning of IR cDNA and the IR-gene promoter, the regulation of the IR gene during differentiation and by various hormones can be studied. Muscle is a major target tissue for insulin action. BC3H1 cells, a mouse muscle cell line in culture, are a model cell type for studying insulin action. Differentiation in these cells results in a 5- to 10-fold increase in IR binding and a 5- to 10-fold increase in IR content. Studies of IR mRNA by Northern and slot-blot analyses reveal a 10-fold increase in IR mRNA after differentiation. These studies indicate that there is a selective increase in IR-gene expression during muscle differentiation. A similar increase in IR-gene expression is observed for the IR during pancreatic acinar cell differentiation. Glucocorticoids increase IR content in several target tissues. Studies in cultured IM-9 lymphocytes indicate that glucocorticoids induce a 5-fold increase in IR mRNA levels. Studies of IR mRNA half-life indicate that glucocorticoids do not alter IR mRNA stability. When the transcription of the IR is measured by elongation assays, glucocorticoids directly stimulate IR transcription 5- to 10-fold. The effect is detectable within 30 min of glucocorticoid treatment and is maximal within 2 h. Therefore, these studies demonstrate that the IR gene is under the direct regulation of glucocorticoids. Insulin downregulates the IR in various target tissues. Prior studies indicate that this downregulation was partly because of accelerated IR degradation. Studying AR42J pancreatic acinar cells, we also found that insulin accelerates IR degradation. Moreover, in these cells, insulin decreases IR biosynthesis by approximately 50%. Studies of IR mRNA indicate there is a concomitant decrease in IR mRNA levels after insulin treatment. Thus, insulin decreases IR-gene expression. The genomic structure of the IR promoter has been elucidated. Primer extension and nuclease S1 analysis indicate that IR mRNA has multiple start sites. The promoter fragment was ligated to a promoterless "reporter" plasmid containing the bacterial gene chloramphenicol acetyltransferase (CAT). When this plasmid is transfected into cultured cells, CAT activity is detected, indicating promoter activity. Various portions of a genomic fragment were ligated to a promoter to study glucocorticoid regulation of the IR promoter. These studies indicate that IR-gene expression is regulated by differentiation and hormonal agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Monoclonal antibodies to the human insulin receptor mimic a spectrum of biological effects in transfected 3T3/HIR fibroblasts without activating receptor kinase.

The effects of four monoclonal antibodies to the alpha subunit of the human insulin receptor were studied in transfected mouse 3T3 fibroblasts expressing human insulin receptors (3T3/HIR). Three antibodies, MA-5, MA-20, and MA-51, mimicked insulin stimulation of the uptake of both 2-deoxy-D-glucose and alpha-aminoisobutyrio acid, and S6 kinase activity. Antibody MA-5 also mimicked insulin stimulation of [3H]thymidine incorporation and cell growth. Although these antibodies mimicked insulin stimulation of biological effects, they failed to significantly activate insulin receptor tyrosine kinase activity. These studies suggest, therefore, that the insulin receptor can signal a variety of cellular functions without stimulation of receptor kinase activity.

Aminoisobutyric Acids↗
PubMed1989

Endocytosis, recycling, and degradation of the insulin receptor. Studies with monoclonal antireceptor antibodies that do not activate receptor kinase.

The endocytosis, recycling, and degradation of the insulin receptor were studied in IM-9 cells and U-937 cells by employing two monoclonal antibodies directed at the alpha subunit of the human insulin receptor, antibodies MA-5 and MA-10. Antibody MA-5 is an insulin agonist and MA-10 is an insulin antagonist (Forsayeth, J., Caro, J.F., Sinha, M.K., Maddux, B.A., and Goldfine, I.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3448-3451). Both monoclonal antibodies, like insulin, induced the endocytosis of the insulin receptor within 15 min. Upon removal of extracellular ligand the internalized receptor recycled to the cell surface. At this time there was no degradation of the receptor as measured by a sensitive insulin receptor radioimmunoassay. After 20 h of incubation, insulin and MA-5, but not MA-10, induced significant receptor degradation as measured by both insulin receptor radioimmunoassay and metabolic labeling studies. These studies demonstrated, therefore, that: 1) internalization and recycling of the receptor can be induced by antireceptor monoclonal antibodies that are either insulin agonists or insulin antagonists; 2) enhanced receptor degradation can be induced by monoclonal antibodies that are insulin agonists; and 3) the process of receptor internalization does not necessarily lead to enhanced receptor degradation. Since prior studies have indicated that neither MA-5 nor MA-10 enhance insulin receptor kinase activity, the present studies also suggest that insulin receptor endocytosis and degradation induced by ligands different than insulin can occur without activation of this process.

Antibodies, Monoclonal↗