Angelo Fusco

Insulin receptor substrate (IRS) transduction system: distinct and overlapping signaling potential.

Insulin receptor substrate (IRS) proteins play a central role in maintaining basic cellular functions such as growth and metabolism. They act as an interface between multiple growth factor receptors possessing tyrosine kinase activity, such as the insulin receptor, and a complex network of intracellular signalling molecules containing Src homology 2 (SH2) domains. Four members (IRS‐1, IRS‐2, IRS‐3, IRS‐4) of this family have been identified which differ in their subcellular distribution and interaction with SH2 domain proteins. In addition, differential IRS tissue‐ and developmental‐specific expression patterns may contribute to specificity in their signaling potential. Copyright

DISTRIBUTION OF INSULIN/INSULIN-LIKE GROWTH FACTOR-I HYBRID RECEPTORS IN HUMAN TISSUES

Insulin receptors (IR) and type 1 IGF receptors (IGF-IR) have been shown to form insulin/IGF-I hybrid receptors in tissues expressing both molecules. The biological function of hybrid receptors is still undefined. To date there is no information about the distribution of hybrid receptors in human tissues. We have applied two microwell-based immunoassays which are capable of quantitating hybrid receptors in small samples of human tissues and cells. Results demonstrated that the proportion of total IGF-IR assembled as hybrids varied between 40 and 60%, thus indicating that hybrid receptors account for a large fraction of total IGF-I binding in human tissues. A significant fraction of total IR was assembled as hybrids in the tissues examined, varying from 37% in placenta to 45% in hepatoma, with the exception of adipose tissue where the fraction of insulin receptors forming hybrids was 17%. Because hybrid receptors bind IGF-I, but not insulin, with high affinity, it is likely that in human tissues hybrid receptors may be primarily activated by IGF-I rather than insulin under physiological conditions. Therefore, differences in hybrid receptors distribution may contribute to regulate tissue sensitivity to insulin and IGF-I by sequestering insulin receptor alphabeta-heterodimer in an IGF-I responsive form.

Erythrocyte Insulin Receptors in Non-insulin-dependent Diabetes Mellitus

Insulin binding was studied on circulating erythrocytes isolated from 12 normal and 12 untreated, adult, nonobese, nonketotic, non-insulin-dependent diabetic subjects. Insulin binding in normal subjects was higher than in diabetics (P < 0.01); binding variation was caused mainly by a reduction in insulin receptor concentration. Insulin binding was inversely correlated with fasting serum insulin levels (R = 0.49; P < 0.01). The close agreement between the present data and previous data on other cell populations suggests that isolated erythrocytes may be a useful tool in clinical investigations on the human insulin receptor.

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, RIN 1046-38 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 long-term 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.

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.

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.

Tissue-specific expression of two alternatively spliced isoforms of the human insulin receptor protein.

Two insulin receptor mRNA species are expressed in human tissues as a result of alternative splicing of exon 11. This event is regulated in a tissue-specific manner. To date, there is little information about the relative abundance of the two receptor protein isoforms on the cell surface. The aim of the present investigation was to assess whether the tissue-specific expression of the two insulin receptor mRNA species is paralleled by a similar pattern of expression of the two receptor protein isoforms. To this end, we assessed the relative distribution of the two receptor variants in various human tissues at the mRNA and protein levels. A PCR-based technique was used to measure the relative abundance of the two mRNA species, and two immunological assays were used to measure the relative steady-state expression of the two receptor protein isoforms. The expression of the two insulin receptor protein isoforms followed the tissue-specific pattern of expression of the two mRNA species.

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

SummaryInsulin-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‡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‡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‡C, acid washing cells, and re-incubating in insulin-free buffer at 37‡C. The radioactivity released by cells was characterized by trichloroacetic acid precipitability, Sephadex G-50 column Chromatograph, and rebinding 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.

Quantitative analysis of pancreatic glucokinase gene expression in cultured β 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.

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.