Increased expression of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of noninsulin-dependent diabetes mellitus subjects.

Insulin receptors (IR) and IGF-I receptors (IGF-IR) have been shown to form hybrid receptors in tissues coexpressing both molecules. To date there is no information about the distribution of hybrids in tissues of normal or diabetic subjects. We developed a microwell-based immunoassay to quantitate hybrids in small human tissues samples. Microwells were coated with MA-20 anti-IR antibody or alpha-IGF-IR-PA antibody directed against the IGF-IR alpha-subunit, and incubated with skeletal muscle extracts of patients with noninsulin-dependent diabetes mellitus (NIDDM) and normal controls. Immobilized receptors were incubated with 125I-insulin or 125I-IGF-I in the presence or absence of the two unlabeled ligands. Hybrids were quantified as the fraction of 125I-IGF-I binding immunoadsorbed with MA-20 and expressed as percentage of total IGF-IR (type I+hybrids) immobilized with alpha-IGF-IR-PA. The immunoassay was validated using Western blotting analysis. Relative abundance of hybrids detected in NIDDM patients was higher than in controls. The percentage of hybrids was negatively correlated with IR number and in vivo insulin sensitivity measured by an insulin tolerance test, whereas the percentage was positively correlated with insulinemia. Insulin binding affinity was lower in NIDDM patients than in controls, and was correlated with the percentage of hybrids. Maximal IGF-I binding was significantly higher in muscle from NIDDM patients compared to controls and was positively correlated with the percentage of hybrid receptors whereas IGF-I binding affinity did not differ between the two groups. These results raise the possibility that alterations in expression of hybrid receptors may contribute to decreased insulin sensitivity, and to increased sensitivity to IGF-I. Because IGF-I has been proposed as a hypoglycemic agent in NIDDM, these results are relevant to the development of new approaches to the treatment of insulin resistance of NIDDM.

The Gly972-->Arg amino acid polymorphism in IRS-1 impairs insulin secretion in pancreatic beta cells.

Recent studies have identified several polymorphisms in the human insulin receptor substrate-1 (IRS-1) gene. The most prevalent IRS-1 variant, a Gly-->Arg change at the codon 972, has been reported to be increased in prevalence among patients with type 2 diabetes. Carriers of the Arg(972) substitution are characterized by lower fasting insulin and C-peptide levels compared with non-carriers, suggesting that the Arg(972) IRS-1 variant may contribute to impairment of insulin secretion. In this study, we stably overexpressed both wild-type IRS-1 (RIN-WT) and Arg(972) IRS-1 variant (RIN-Arg(972)) in RIN beta cells to investigate directly whether the polymorphism in codon 972 of IRS-1 impairs insulin secretion. The Arg(972) IRS-1 variant did not affect expression or function of endogenous IRS-2. RIN-WT showed a marked increase in both glucose- and insulin-stimulated tyrosine phosphorylation of IRS-1 compared with control RIN cells. The Arg(972) IRS-1 variant did not alter the extent of either glucose- or insulin-stimulated tyrosine phosphorylation of recombinant IRS-1. However, RIN-Arg(972) showed a significant decrease in binding of the p85 subunit of phosphatidylinositol-3-kinase (PI 3-kinase) with IRS-1, compared with RIN-WT. Compared with control RIN cells, insulin content was reduced to the same extent in RIN-WT or RIN-Arg(972) at both the protein and mRNA levels. Both glucose- and sulfonylurea-induced insulin secretion was increased in RIN-WT compared with control RIN cells. By contrast, RIN cells expressing Arg(972) IRS-1 exhibited a marked decrease in both glucose- and sulfonylurea-stimulated insulin secretion compared with RIN-WT. These data suggest that the insulin signaling pathway involving the IRS-1/PI 3-kinase may play an important role in the insulin secretory process in pancreatic beta cells. More importantly, the results suggest that the common Arg(972) IRS-1 polymorphism may impair glucose-stimulated insulin secretion, thus contributing to the relative insulin deficiency observed in carriers of this variant.

Tissue-specific antibodies against the fibroblast insulin receptor in a patient with lupus nephritis and hypoglycemia.

We recently reported that the serum from a patient with lupus nephritis, insulin resistance, and hypoglycemia contains multiple populations of antibodies directed at the human insulin receptor. In the present study, we found a subpopulation of antibodies (eluted from a protein A-Sepharose affinity column at pH 4.3) directed at the human fibroblast insulin receptor. When tested against human placental membranes, IM-9 lymphocytes, circulating monocytes and erythrocytes, and isolated adipocytes, the antibody subpopulation did not compete with 125I-insulin for binding to its receptor. In contrast, the antibody subpopulation competed with 125I-insulin for binding to the human fibroblast insulin receptor. This antibody subpopulation stimulated [3H]alpha-aminoisobutyric acid [( 3H]AIB) uptake to these cells. Unlike the effect of insulin, however, this regulation of transport was not antagonized by a mouse monoclonal antibody to the human insulin receptor that inhibits 125I-insulin binding. These studies indicate, therefore, that a tissue-specific antibody subpopulation can occur spontaneously in patients with antibodies to the human insulin receptor. Furthermore, they indicate the presence of anti-insulin receptor autoantibodies specifically directed against a tissue that is not primarily involved in glucose metabolism.

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)

Expression, processing, and secretion of the neuroendocrine VGF peptides by INS-1 cells.

The neurotropin-inducible gene vgf is expressed in neuronal and endocrine tissues. It encodes a secretory protein that is proteolytically processed in neuronal cells to low molecular mass polypeptides. In the present report, we show that vgf is expressed in different insulinoma cell lines and in normal rat pancreatic islets. In the insulinoma-derived beta-cell line INS-1, vgf messenger RNA was transcriptionally up-regulated by increased levels ofintracellular cAMP, but not by the addition of glucose (20 mM) or phorbol 12-myristate 13-acetate (100 nM). Furthermore, nerve growth factor failed to stimulate vgf gene expression. In INS-1 cells, the VGF protein was shown to be processed in a post endoplasmic reticulum compartment to produce a peptide profile similar to that seen in neurons. The release of such VGF peptides occurred at a low rate in the absence of secretory stimuli (<2%/h). A 3-fold increase in the rate of release was seen after the addition of glucose (15 mM), a 4-fold increase was seen after (Bu)2cAMP (1 mM), and a 6-fold increase was seen after phorbol 12-myristate 13-acetate (100 nM). These results indicated that insulin-containing cells produce VGF-derived peptides that are released via a regulated pathway in response to insulin secretagogues.

Molecular and functional characterization of pituitary adenylate cyclase-activating polypeptide (PACAP-38)/vasoactive intestinal polypeptide receptors in pancreatic beta-cells and effects of PACAP-38 on components of the insulin secretory system.

It has been previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) regulates insulin secretion. PACAP exerts its biological action by binding to at least three different receptor subtypes coupled to different signal transduction mechanisms. The signaling pathways underlying the insulinotropic effect of PACAP involve mainly the activation of adenylate cyclase to form cAMP, which directly and indirectly, through increased intracellular Ca2+, stimulates insulin exocytosis. In the present study we have characterized the functional and molecular expression of PACAP/vasoactive intestinal polypeptide receptors isoforms and subtypes and its isoforms in a beta-cell line and in isolated rat pancreatic islets. Although insulinoma cells express the messenger RNA encoding PAC1 (-R and -hop variants), VPAC1 and VPAC2, binding experiments indicate the preponderance of PAC1 over VPAC 1-2 receptors. We have also shown that the main signaling pathway of PACAP in beta-cells is mediated by adenylate cyclase, whereas the inositol 1,4,5-trisphosphate pathway is almost inactive. Furthermore, we have demonstrated that PACAP exerts long-term effects on beta-cells, such as transcriptional regulation of the insulin gene and genes of the glucose-sensing system (GLUT1 and hexokinase 1).

The Gly-->Arg972 amino acid polymorphism in insulin receptor substrate-1 affects glucose metabolism in skeletal muscle cells.

Molecular scanning of insulin receptor substrate-1 (IRS-1) revealed several amino acid substitutions. The most common IRS-1 variant, a Gly to Arg972 change, is more prevalent among type 2 diabetic patients. In this study we overexpressed wild-type and Arg972IRS-1 variant in L6 skeletal muscle cells and examined the functional consequences of this polymorphism on insulin metabolic signaling. L6 cells expressing Arg972-IRS-1 (L6-Arg972) showed a decrease in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity compared with L6 cells expressing wild-type IRS-1 (L6-WT) as a consequence of decreased binding of p85 subunit of PI 3-kinase to IRS-1. L6-Arg972 exhibited a decrease in both basal and insulin-stimulated glucose transport due to a reduction in the amount of both GLUT1 and GLUT4 translocated to the plasma membrane. Both basal and insulin-stimulated Akt phosphorylations were decreased in L6-Arg972 compared with L6-WT. Basal glycogen synthase kinase-3 (GSK-3) activity was increased in L6-Arg972 compared with L6-WT, and insulin-induced inactivation of GSK-3 was also reduced in L6-Arg972. This change was associated with a significant decrease in insulin-stimulated glucose incorporation into glycogen and glycogen synthase activity in L6-Arg972 compared with L6-WT. These results indicate that the Arg972-IRS-1 polymorphism impairs the ability of insulin to stimulate glucose transport, glucose transporter translocation, and glycogen synthesis by affecting the PI 3-kinase/Akt/GSK-3 signaling pathway. The present data indicate that the polymorphism at codon 972 of IRS-1 may contribute to the in vivo insulin resistance observed in carriers of this variant.

Increased abundance of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of obese subjects is correlated with in vivo insulin sensitivity.

We reported that in noninsulin-dependent diabetes melitus (NIDDM) patients expression of insulin/insulin-like growth factor I (IGF-I) hybrid receptors is increased in insulin target tissues. Whether this is a defect associated with NIDDM or represents a generalized abnormality associated with insulin resistant states is still unsettled. To address this, we applied a microwell-based immunoassay to measure abundance of insulin receptors, type 1 IGF receptors, and hybrid receptors in muscle of eight normal and eight obese subjects. Maximal insulin binding to insulin receptors was lower in obese than in control subjects (B/T = 1.8 +/- 0.20 and 2.6 +/- 0.30; P < 0.03, respectively) and was negatively correlated with insulinemia (r = -0.60; P < 0.01). Maximal IGF-I binding to type 1 IGF receptors was higher in obese than in controls (B/T = 1.9 +/- 0.20 and 0.86 +/- 0.10; P < 0.0001, respectively) and was negatively correlated with plasma IGF-I levels (r = -0.69; P < 0.003). Hybrid receptor abundance was higher in obese than in normal subjects (B/T = 1.21 +/- 0.14 and 0.44 +/- 0.06; P < 0.0003, respectively) and was negatively correlated with insulin binding (r = -0.60; P < 0.01) and positively correlated with IGF-I binding (r = 0.92; P < 0.0001). Increased abundance of hybrids was correlated with insulinemia (r = 0.70; P < 0.002) and body mass index (r = 0.71; P < 0.0019), whereas it was negatively correlated with in vivo insulin sensitivity measured by ITT (r = -0.67; P < 0.016). These results indicate that downregulation of insulin receptors or upregulation of type 1 IGF receptors because of changes in plasma insulin and IGF-I levels may result in modifications in hybrid receptor abundance.

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.

The effects of pregnancy steroids on adaptation of beta cells to pregnancy involve the pancreatic glucose sensor glucokinase.

Pregnancy is associated with adaptive changes including increased number and size of beta cells and enhanced gap-junctional coupling among beta cells, increased glucose-induced insulin response and decreased glucose stimulation threshold. The role exerted by pregnancy steroids and lactogenic hormones in the development of islets upregulation during pregnancy has been widely investigated. In the present study we studied the possibility that pregnancy steroids induce functional modifications of beta cells involving the expression and function of glucokinase. Our results indicate that estradiol and progesterone do not influence significantly glucokinase mRNA expression, while they induce a dose-dependent and time-dependent increase of glucokinase activity in RIN 1046-38 cells. The increased enzymatic activity results in an increased glucose-induced insulin release. Therefore it is possible to hypothesize that pregnancy steroids influence glucokinase expression in beta cells at a post-transcriptional level and that this effect contributes to the development of hyperinsulinemia during pregnancy.

Distribution of adrenocorticoid receptors in the rat CNS measured by competitive PCR and cytosolic binding.

Combined quantitative polymerase chain reaction (PCR) and cytosolic binding assay techniques are used to measure mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA, Kd, and Bmax in various rat central nervous system (CNS) regions, namely amygdala, hypothalamus, hippocampus, cortex, pituitary, and cervical, thoracic, and lumbar spinal cord. Two internal standards (i.s.) cDNA were cloned for quantitative PCR purposes. The i.s. templates differed from the respective wild-type (wt) templates for a single base-pair mutation introduced by PCR that generated a unique restriction site, thus allowing amplification products arising from coamplification of wt and i.s. to be distinguished. Results show that cerebellum, which displayed average Bmax values for both receptors, contained the highest level of MR and GR mRNA. Hippocampus also had a high level of MR mRNA. Low mRNA content was found in the hypothalamus for MR and GR as well as in the cortex for GR. High Bmax values for both MR and GR were found in the lumbar spinal cord, despite a modest mRNA content. The lowest Bmax values were found in the cortex for both receptors. It is, therefore, concluded that mRNA content and Bmax are not closely correlated in the rat CNS. These data suggest a differential regulation of various adrenocorticoid receptor isoforms. Moreover, this quantitative PCR method is very sensitive and can be used to assay small amounts of material in order to obtain absolute measurements of mRNA expression.

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.

Corticosterone treatment differentially affects adrenocorticoid receptors expression and binding in the hippocampus and spinal cord of the rat.

We have studied the immediate and long-term effects of high doses of corticosterone (CORT) on mRNA expression and binding properties of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and spinal cord of rats. Animals were treated with corticosterone (10 mg/d subcutaneously) for 21 consecutive days, and mineralocorticoid and glucocorticoid receptors were studied either 24 h or 2 wk after the last injection. Major results show that corticosterone treatment reduces mineralocorticoid and glucocorticoid receptor maximum binding capacity (Bmax) in both the hippocampus and spinal cord and that this reduction is partially reversed after cessation of treatment. With respect to mRNA expression, in the hippocampus recovery after cessation of treatment is complete. By contrast, in the spinal cord, mineralocorticoid receptor mRNA expression is irreversibly increased after treatment, but the glucocorticoid receptor mRNA level remains unaffected during and after treatment. Thus, these data suggest the presence of distinct regulatory mechanisms for adrenocorticoid receptors in rat brain and spinal cord, in response to long-term exposure to high levels of circulating corticosterone and after recovery from treatment.

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.

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.

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.

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.

Increased abundance of insulin/IGF-I hybrid receptors in adipose tissue from NIDDM patients.

Insulin/IGF-I hybrid receptors composed of an insulin receptor (IR) alphabeta-hemireceptor and a type 1 IGF receptor (IGF-IR) alphabeta-hemireceptor are formed in tissues expressing both molecules. To date there is a limited information about the proportion of hybrids in tissues of normal or diabetic subjects. In this study, we determined the abundance of hybrids in fat from control and NIDDM subjects by using a microwell-based immunoassay. Microwells coated with MA-20 anti-IR or alpha-IGF-IR-PA anti-IGF-IR antibody were incubated with tissue extracts. Immunoadsorbed receptors were incubated with 125I-insulin or 125I-IGF-I in the presence or absence of unlabeled ligands, and hybrids were quantitated as the fraction of 125I-IGF-I binding immunoadsorbed with MA-20. Abundance of hybrids was increased in NIDDM patients as compared with controls (B/T = 1.29 +/- 0.18 and 0.52 +/- 0.06%; P < 0.008, respectively), and it was inversely correlated with both IR number (r = -0.65; P < 0.002), and in vivo insulin sensitivity measured by insulin tolerance test (r = -0.75; P < 0.005), whereas it was positively correlated with insulinemia (r = 0.63; P < 0.003). Insulin binding affinity was lower in NIDDM subjects than in controls (ED50 = 1.87 +/- 0.32 and 0.54 +/- 0.20 nmol/l; P < 0.009, respectively), and was correlated with the percentage of hybrids. Maximal IGF-I binding was significantly greater in NIDDM patients than controls and was positively correlated with the percentage of hybrids whereas IGF-I binding affinity did not differ between the two groups. Results show that expression of hybrids is increased in fat of NIDDM patients compared to control subjects and is correlated with in vivo insulin sensitivity thus raising the possibility that alterations in expression of hybrids which bind IGF-I with higher affinity than insulin may contribute, at least in part, to insulin resistance.

GLUT2 and glucokinase expression is coordinately regulated by sulfonylurea.

In the present study we examined the effect of sulfonylurea on the expression of the glucose transporter GLUT2 and the glucose phosphorylating enzyme Glucokinase (GK) in betaTC6-F7 cells; furthermore, we studied the modifications induced by sulfonylurea on glucose-responsiveness and -sensitivity. Results demonstrate that sulfonylurea increases GLUT2 and GK mRNA expression after 24 h in a dose-dependent manner. On the contrary, after 48 and 72 h a time-dependent reduction of both GLUT2 and GK mRNA occurs. GLUT2 and GK protein expression follow the same modifications. Therefore, GLUT2 and GK are coordinately regulated by sulfonylurea, probably by a common mechanism. Glucose-induced insulin release is increased by sulfonylurea as well as glucose sensitivity. Our study suggests that short-term effect of sulfonylurea increases while long-term effect reduces the expression of glucose sensing elements. The long-term inhibitory effect on glucose sensing elements would explain the reduced insulin secretion occurring after chronic sulfonylurea treatment.

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.