The role of Humphrey Matrix testing in the early diagnosis of retinopathy in type 1 diabetes.

AIM: The aim of the study was to investigate the role of Humphrey Matrix threshold testing in the detection of early functional retinal impairment in subjects with type 1 diabetes mellitus (DM1) without any signs of retinal vasculopathy, and to investigate the relationship between both functional and structural retinal parameters and metabolic control. METHODS: Thirty eyes of 30 subjects with DM1, with no sign of retinal vasculopathy, and 30 eyes of 30 age- and sex-matched healthy subjects were enrolled in this cross-sectional clinical study. Functional testing included Humphrey Matrix perimetry and white-on-white Humphrey visual field perimetry (HFA), while retinal nerve fibre layer (RNFL) thickness was measured by scanning laser polarimetry with variable corneal birefringence compensator (GDx VCC). RESULTS: Matrix mean deviation (MD) was found to be significantly reduced in subjects with DM1 compared with controls (-1.10 (SD 2.98; 95% CI -2.21 to 0.01) vs 1.37 (SD 2.11; 95% CI 0.58 to 2.16), p = 0.0005). HFA MD and pattern standard deviation (PSD) were significantly worse in subjects with DM1 compared with controls (p = 0.010 and p = 0.013 respectively). Among structural parameters, average peripapillary RNFL thickness was reduced in DM1 subjects (p = 0.006). Matrix MD and HFA MD and PSD, and average peripapillary and superior RNFL, were significantly reduced in subjects with DM1 with HbA(1c) > or = 7% compared with controls. CONCLUSIONS: Functional and structural retinal testing by Humphrey Matrix and GDx VCC could be useful for the identification of early retinal impairment in people with DM1 with no sign of retinal vasculopathy.

NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro.

AIMS/HYPOTHESIS: Using primary cultures of human pancreatic islets, purified human pancreatic beta cells and the mouse beta TC6-F7 cell line, we analysed the expression of nerve growth factor, (NGF/NGF) receptors in beta cells. To investigate whether NGF could sub-serve an autocrine antiapoptotic role in beta cells, we studied the effects of NGF withdrawal using a neutralizing monoclonal anti-NGF antibody. METHODS: The expression of NGF and NGF receptors (gp140(Trk-A) and p75(NTR)) were analysed by RT-PCR and immunofluorescence. Pulse-chase experiments and beta cell/PC12 co-cultures were used to investigate NGF production and secretion from beta cells. Possible apoptosis induced by NGF withdrawal was monitored by phosphatidylserine translocation, nucleosomal formation, DNA laddering and FACS analysis. Involvement of transcription/translation mechanisms were investigated as well as the gp140(Trk-A) required. Finally, signal transduction pathways typically involved in apoptotic mechanisms were analysed by western blot analysis. RESULTS: We show that NGF and both NGF receptors, gp140(Trk-A) and p75(NTR) are expressed in beta cells where NGF is produced and secreted in a biologically active form. NGF-withdrawal induces beta-cell transcription/translation independent apoptosis but mediated by gp140(Trk-A). Analysis of signal transduction pathways revealed that NGF withdrawal inhibits the PI3-K, protein kinase B (AKT), Bad survival pathway and activates c-Jun kinase (JNK) whereas ERKs and p38 mitogen-activated protein kinase (MAPK) are not affected. Moreover, Bcl-XL, but not Bcl-2 protein expression are reduced. CONCLUSION/INTERPRETATION: We suggest that the integrity of the NGF/NGF receptor system and NGF bioavailability participate in controlling beta-cell survival in culture which represents a key issue for improving possibilities for transplantations in the treatment of diabetes.

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance.

AIMS/HYPOTHESIS: Leptin resistance in obese humans seems to be predominantly caused by signalling abnormalities at the post receptor level. Leptin resistance in obese individuals is frequently associated with insulin resistance and pronounced hyperinsulinaemia indicating a negative crosstalk of the insulin and leptin signalling chain. METHODS: This hypothesis was tested using a cell model of peripheral leptin signalling, i. e. insulin-secreting cell lines (RINr1046-38). Mechanisms for a crosstalk between the insulin and leptin signalling pathway were also studied in rat-1 and HEK293 cells overexpressing elements of the insulin and leptin signalling chain. RESULTS: The effects of leptin on insulin secretion are completely cancelled by a 4-h preincubation with 1 nmol/l insulin, supporting the hypothesis of a negative crosstalk of insulin and leptin signalling. We investigated the potential molecular mechanisms in more detail in HEK293 cells and Rat-1 fibroblasts that overexpressed proteins of the insulin and leptin signalling chain. Leptin (60 ng/ml) stimulated autophosphorylation of JAK-2 in HEK 293 cells. This leptin effect could be inhibited by simultaneous treatment of cells with insulin. Furthermore, overexpression of the insulin receptor in HEK 293 cells clearly reduced JAK-2 phosphorylation and led further downstream to a diminished phosphatidylinositol 3-kinase activity. The inhibitory effect of the insulin signal could be partially prevented by transfection of the cells with an inactive mutant of the tyrosine phosphatase SHP-1. CONCLUSION/INTERPRETATION: In summary, our data suggest that the insulin receptor signalling pathway interferes with leptin signalling at the level of JAK-2. Inhibition of JAK-2 phosphorylation might occur through SHP-1-dependent pathways, indicating that hyperinsulinaemia contributes to the pathogenesis of leptin resistance.

The sulfonylurea glimepiride regulates intracellular routing of the insulin-receptor complexes through their interaction with specific protein kinase C isoforms.

Sulfonylureas may stimulate glucose metabolism by protein kinase C (PKC) activation. Because interaction of insulin receptors with PKC plays an important role in controlling the intracellular sorting of the insulin-receptor complex, we investigated the possibility that the sulfonylurea glimepiride may influence intracellular routing of insulin and its receptor through a mechanism involving PKC, and that changes in these processes may be associated with improved insulin action. Using human hepatoma Hep-G2 cells, we found that glimepiride did not affect insulin binding, insulin receptor isoform expression, and insulin-induced receptor internalization. By contrast, glimepiride significantly increased intracellular dissociation of the insulin-receptor complex, degradation of insulin, recycling of internalized insulin receptors, release of internalized radioactivity, and prevented insulin-induced receptor down-regulation. Association of PKC-betaII and -epsilon with insulin receptors was increased in glimepiride-treated cells. Selective depletion of cellular PKC-betaII and -epsilon by exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment of cells with PKC-betaII inhibitor G06976 reversed the effect of glimepiride on intracellular insulin-receptor processing. Glimepiride increased the effects of insulin on glucose incorporation into glycogen by enhancing both sensitivity and maximal efficacy of insulin. Exposing cells to TPA or G06976 inhibitor reversed these effects. Results indicate that glimepiride increases intracellular sorting of the insulin-receptor complex toward the degradative route, which is associated with both an increased association of the insulin receptor with PKCs and improved insulin action. These data suggest a novel mechanism of action of sulfonylurea, which may have a therapeutic impact on the treatment of type 2 diabetes.

The common Arg972 polymorphism in insulin receptor substrate-1 causes apoptosis of human pancreatic islets.

Molecular scanning of human IRS-1 gene revealed a common polymorphism causing Gly-->Arg972 change. Diabetic and pre-diabetic carriers of Arg972 IRS-1 are characterized by low fasting levels of insulin and C-peptide. To investigate directly whether the Arg 972 IRS-1 affects human islet cells survival, we took advantage of the unique opportunity to analyze pancreatic islets isolated from three donors heterozygous for the Arg972 and six donors carrying wild-type IRS-1. Islets from carriers of Arg972 IRS-1 showed a two-fold increase in the number of apoptotic cells as compared with wild-type. IRS-1-associated PI3-kinase activity was decreased in islets from carriers of Arg972 IRS-1. Same results were reproduced in RIN rat b-cell lines stably expressing wild-type IRS-1 or Arg972 IRS-1. Using these cells, we characterized the downstream pathway by which Arg972 IRS-1 impairs b-cell survival. RIN-Arg972 cells exhibited a marked impairment in the sequential activation of PI3-kinase, Akt, and BAD as compared with RI N-WT. Impaired BAD phosphorylation resulted in increased binding to Bcl-XL instead of 14-3-3 protein, thus sequestering the Bcl-XL antiapoptotic protein to promote survival. Both caspase-9 and caspase-3 activities were increased in RIN-Arg972 cells. The results show that the common Arg972 polymorphism in IRS-1 impairs human b-cell survival and causes resistance to antiapoptotic effects of insulin by affecting the PI3-kinase/Akt survival pathway. These findings establish an important role for the insulin signaling in human b-cell survival and suggest that genetic defects in early steps of insulin signaling may contribute to b-cell failure.

Neurotrophins and neurotrophin receptors mRNAs expression in pancreatic islets and insulinoma cell lines.

CONTEXT: It is worth noting that islets and betaTC6-F7 cells share a common pattern of expression of neurotrophins and neurotrophin receptors. Recently, several studies have hypothesized a role for nerve growth factor in pancreatic development and maturation, suggesting that nerve growth factor may be a survival factor for pancreatic beta-cells. OBJECTIVE: The aim of the present study was to investigate the pattern of expression of neurotrophins and their relative receptors both in rat pancreatic islets and in a wide panel of insulinoma cell lines. MAIN OUTCOME MEASURES: A semi-quantitative reverse-transcription polymerase chain reaction analysis was performed on ribonucleic acids extracted from these cells. RESULTS: Reverse transcription-polymerase chain reaction analysis demonstrates that brain-derived neurotrophic factor, as well as neurotrophins 3 and 4, are expressed both in islets and in all insulinoma cells, while nerve growth factor is expressed only in islets, betaTC6-F7 cells and, at a low level, in RIN 1046-38 cells. Receptors protein tyrosine kinase A and C are ubiquitously expressed both in islets and insulinoma cells. Tyrosine kinase B is absent in HIT-T15 cells. CONCLUSIONS: These data indicate that betaTC6-F7 cells are a suitable model for studying the role of neurotrophins in the survival of beta-cells.

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.

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.

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).

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.

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.

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.

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.

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.

Dose-dependent effect of octreotide on insulin secretion after OGTT in obesity.

OBJECTIVE: The present study aimed at evaluating the acute effect of increasing doses of octreotide (OCT), a long-acting somatostatin analogue, on glucose tolerance and insulin secretion. METHODS: A standard and two other oral glucose tolerance tests 30 min after subcutaneous administration of OCT were performed in randomized order in each subject. Obese subjects received 10, 25, or 50 microg of OCT; control subjects received only 10 and 25 microg. Fifteen obese and 10 control subjects were studied; all of them had a normal glucose tolerance. Plasma glucose and insulin levels were measured at times -30, 0, 30, 60, 90, 120, 150, and 180 min after the glucose tolerance test. RESULTS: The results demonstrated that, following OCT administration, both control and obese subjects developed a reduced glucose tolerance, a delayed glycemic peak, and an increase of late plasma glucose values. Fasting as well as stimulated insulin secretions were higher in obese subjects as compared with controls, and insulin secretion was inhibited in a dose-dependent manner by OCT. CONCLUSIONS: These data indicate that the action of OCT might be due to at least two different cooperative mechanisms: (1) a delayed glucose absorption, as suggested by the delay of glycemic peak, and (2) a direct or vagal-mediated effect on beta-cells, as suggested by the reduction of the area under the curve values in spite of the elevated late glycemic levels. It is noteworthy that doses of OCT as low as 10 and 25 microg are sufficient to inhibit insulin secretion both in normal and obese subjects.

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.

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.

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.

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.