Analysis of circulating osteoclast and osteogenic precursors in patients with Gorham-Stout disease.

PURPOSE: Gorham-Stout disease is a very rare disorder characterized by progressive bone erosion and angiomatous proliferation; its etiopathogenesis is still unknown, and diagnosis is still performed by exclusion criteria. The alteration of bone remodeling activity has been reported in patients; in this study, we characterized circulating osteoclast and osteogenic precursors that could be important to better understand the osteolysis observed in patients. METHODS: Flow cytometry analysis of PBMC (Peripheral Blood Mononuclear Cells) was performed to characterize circulating osteoclast and osteogenic precursors in GSD patients (n = 9) compared to healthy donors (n = 55). Moreover, ELISA assays were assessed to evaluate serum levels of bone markers including RANK-L (Receptor activator of NF-κB ligand), OPG (Osteoprotegerin), BALP (Bone Alkaline Phosphatase) and OCN (Osteocalcin). RESULTS: We found an increase of CD16-/CD14+CD11b+ and CD115+/CD14+CD11b+ osteoclast precursors in GSD patients, with high levels of serum RANK-L that could reflect the increase of bone resorption activity observed in patients. Moreover, no significant alterations were found regarding osteogenic precursors and serum levels of BALP and OCN. CONCLUSION: The analysis of circulating bone cell precursors, as well as of RANK-L, could be relevant as an additional diagnostic tool for these patients and could be exploited for therapeutic purposes.

A clinical, histopathological and laboratory study of 19 consecutive Italian paediatric patients with chilblain-like lesions: lights and shadows on the relationship with COVID-19 infection.

BACKGROUND: Acral chilblain-like lesions are being increasingly reported during COVID-19 pandemic. However, only few patients proved positivity for SARS-CoV-2 infection. The relationship between this skin manifestation and COVID-19 infection has not been clarified yet. OBJECTIVE: To thoroughly characterize a prospective group of patients with chilblain-like lesions and to investigate the possible relationship with SARS-CoV-2 infection. METHODS: Following informed consent, patients underwent (i) clinical evaluation, (ii) RT-PCR and serology testing for SARS-CoV-2, (iii) digital videocapillaroscopy of finger and toe nailfolds, (iv) blood testing to screen for autoimmune diseases and coagulation anomalies, and (v) skin biopsy for histopathology, direct immunofluorescence and, in selected cases, electron microscopy. RESULTS: Nineteen patients, all adolescents (mean age: 14 years), were recruited. 11/19 (58%) of them and/or their cohabitants reported flu-like symptoms one to two months prior to skin manifestation onset. Lesions were localized to toes and also heels and soles. Videocapillaroscopy showed pericapillary oedema, dilated and abnormal capillaries, and microhaemorrhages both in finger and toe in the majority of patients. Major pathological findings included epidermal basal layer vacuolation, papillary dermis oedema and erythrocyte extravasation, perivascular and perieccrine dermal lymphocytic infiltrate, and mucin deposition in the dermis and hypodermis; dermal vessel thrombi were observed in two cases. Blood examinations were normal. Nasopharyngeal swab for SARS-CoV-2 and IgG serology for SARS-CoV-2 nucleocapsid protein were negative. Importantly, IgA serology for S1 domain of SARS-CoV-2 spike protein was positive in 6 patients and borderline in 3. CONCLUSIONS: Chilblain-like lesions during COVID-19 pandemic have specific epidemiologic, clinical, capillaroscopic and histopathological characteristics, which distinguish them from idiopathic perniosis. Though we could not formally prove SARS-CoV-2 infection in our patients, history data and the detection of anti-SARS-COV-2 IgA strongly suggest a relationship between skin lesions and COVID-19. Further investigations on the mechanisms of SARS-CoV-2 infection in children and pathogenesis of chilblain-like lesions are warranted.

Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

Missense mutations in the TGM2 gene encoding transglutaminase 2 are found in patients with early-onset type 2 diabetes. Mutation in brief no. 982. Online.

Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2-/- Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic beta cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion.

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.

Divergent phenotype of two siblings human leukocyte antigen identical, affected by nonclassical and classical congenital adrenal hyperplasia caused by 21-hydroxylase deficiency.

CONTEXT: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders most often caused by enzyme 21-hydroxylase deficiency. Most mutations causing enzymatic deficiency are generated by recombinations between the active gene CYP21 and the pseudogene CYP21P. Only 1-2% of affected alleles result from spontaneous mutations. The phenotype of CAH varies greatly, usually classified as classical or nonclassical, depending on variable degree in 21-hydroxylase activity. Here we report a divergent phenotype of two human leukocyte antigen identical siblings, affected by nonclassical and classical CAH caused by 21-hydroxylase deficiency due to different genotype. PATIENTS AND METHODS: Using direct sequencing method and Southern blot, we studied two children (one male and one female), affected, respectively, by nonclassical and classical CAH and their parents. RESULTS: The mother was heterozygous for the Q318X mutation, and the father was heterozygous for the V281L mutation. The brother was a compound heterozygote for the mutations V281L and Q318X, whereas the proband was compound heterozygote for the Q318X mutation and a large conversion. The two children are human leukocyte antigen identical (A*02;B*14;DRB1*01/A*33;B*14;DRB1*03). CONCLUSIONS: Different phenotype of the proband is the result of compound heterozygosity for the maternal mutation Q318X and a de novo large conversion.

Expression of insulin/IGF-I hybrid receptors is increased in skeletal muscle of patients with chronic primary hyperinsulinemia.

The insulin receptor (IR) shares structural and functional homology with the IGF-I receptor (IGF-IR). Hybrid receptors composed of an IR alphabeta-heterodimer and an IGF-IR alphabeta-heterodimer are formed in tissues expressing both molecules. Hybrids behave as IGF-IR rather than IR with respect to ligand binding affinity, receptor autophosphorylation, and hormone internalization and degradation. Factors regulating hybrid formation in vivo are unknown. We recently reported that in skeletal muscle of NIDDM patients, expression of hybrids is increased and correlated with a decrease in IR number and an increase in fasting insulin levels. However, it is not clear whether increased expression of hybrid receptors is a primary defect specifically associated with NIDDM or a secondary event caused by hyperinsulinemia. To address this issue, we used a quantitative microwell-based immunoassay to measure hybrid receptor abundance in skeletal muscle of 11 normal subjects and 12 patients with insulinoma, a state of primary nongenetically determined hyperinsulinemia. Total insulin binding was lower in insulinoma patients than in normal subjects (0.70 +/- 0.18 vs. 4.59 +/- 0.77; P < 0.0001). Total IGF-I binding did not differ between the two groups (0.81 +/- 0.27 and 0.85 +/- 0.10, respectively). The amount of hybrids, expressed as bound/total (B/T), was higher in patients with insulinoma than in normal subjects (0.57 +/- 0.19 vs. 0.36 +/- 0.03; P < 0.0006) and was inversely correlated with total insulin binding (r = -0.64, P < 0.0004). Increased abundance of hybrid receptors was positively correlated with insulin levels (r = -0.82, P < 0.0009) and inversely correlated with insulin-mediated glucose uptake (r = -0.80, P < 0.01). No correlations were observed between insulin-mediated glucose uptake and maximal specific insulin binding (r = 0.19, P = 0.64). These results indicate that insulin-induced IR downregulation may lead to the formation of a higher proportion of hybrid receptors, whose abundance is negatively correlated with in vivo insulin sensitivity. These results, therefore, support a role for insulin in the regulation of hybrid receptors formation and suggest that increased expression of hybrids in NIDDM may be a secondary event caused by hyperinsulinemia rather than a primary defect.

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.

Somatostatin infusion withdrawal: studies in normal children and in children with growth hormone deficiency.

Withdrawal of a somatostatin infusion (SSIW) is followed by a rebound rise of GH in both animals and normal adult men, a phenomenon likely mediated by endogenous GHRH function. In the present study, we have evaluated the GH response to SSIW in a group of 28 prepubertal children (18 boys and 10 girls; aged 3.7-11.1 yr). Six children had GH deficiency [GHD; GH responses to pyridostigmine (PD)+GHRH and to clonidine <20 and <7 microg/L, respectively], 4 children had GH neurosecretory dysfunction (GHND; GH responses to PD+GHRH and to clonidine > or =20 and >7 microg/L, respectively; mean integrated nighttime GH concentrations <3 microg/L), and 18 children were short normal children [normal controls (NC)]. All children received a constant infusion of SS at the dose of 3 microg/Kg x h for 90 min. SSIW elicited a clear-cut GH rise in NC children (13.7+/-1.0 microg/L), but not in GH-deficient children, regardless of the underlying etiology (GHD, 1.6+/-0.4 microg/L; GHND, 2.4+/-0.3 microg/L). The GH response to SSIW was similar between GHD and GHND children. There was no overlapping of the maximum SSIW-stimulated GH peaks between NC and GHD or GHND children. In conclusion, we have demonstrated that SSIW elicits a significant GH rise in NC children, but not in GH-deficient children, regardless of the underlying etiology (GHD or GHND). This resulted in complete discrimination of NC from GHD or GHND children. Were these present findings confirmed on a larger number of children, SSIW, because of its testing efficaciousness and safety, procedural simplicity, and economy holds promise of being a useful diagnostic tool for GH-dependent growth disorders.

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 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.

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.

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.

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.

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.

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

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