Human proinsulin production in the milk of transgenic cattle.

BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Condensation of the ß-cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain.

ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of ß-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 µm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.

Effects of GLP-1 and GIP on Islet Function in Glucose-Intolerant, Pancreatic-Insufficient Cystic Fibrosis.

Impaired insulin and incretin secretion underlie abnormal glucose tolerance (AGT) in pancreatic insufficient cystic fibrosis (PI-CF). Whether the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) can enhance pancreatic islet function in cystic fibrosis (CF) is not known. We studied 32 adults with PI-CF and AGT randomized to receive either GLP-1 (n = 16) or GIP (n = 16) during glucose-potentiated arginine (GPA) testing of islet function on two occasions, with either incretin or placebo infused, in a randomized, double-blind, cross-over fashion. Another four adults with PI-CF and normal glucose tolerance (NGT) and four matched control participants without CF underwent similar assessment with GIP. In PI-CF with AGT, GLP-1 substantially augmented second-phase insulin secretion but without effect on the acute insulin response to GPA or the proinsulin secretory ratio (PISR), while GIP infusion did not enhance second-phase or GPA-induced insulin secretion but increased the PISR. GIP also did not enhance second-phase insulin in PI-CF with NGT but did so markedly in control participants without CF controls. These data indicate that GLP-1, but not GIP, augments glucose-dependent insulin secretion in PI-CF, supporting the likelihood that GLP-1 agonists could have therapeutic benefit in this population. Understanding loss of GIP's insulinotropic action in PI-CF may lead to novel insights into diabetes pathogenesis.

Novel Flow Cytometric Immunoassay for Detection of Proinsulin Autoantibodies in Diabetes Mellitus Employing a Recombinant Autoantigen Expressed in E. coli.

Introduction: Insulin and proinsulin autoantibodies (IAA/PAA) are usually the first markers to appear in patients with type 1 Diabetes Mellitus (T1DM) and their prevalence ranges from 10 to 60% in the child-adolescent population. The reference method for IAA/PAA detection is the Radioligand Binding Assay (RBA), a highly specific and sensitive technique, but expensive and polluting. The aim of this work was to develop a novel flow cytometric microsphere-based immunoassay (FloCMIA) for PAA detection, employing recombinant human proinsulin (PI), as an alternative method to RBA, less expensive and harmful to the environment. Materials and Methods: Human PI was expressed as Thioredoxin fusion protein (TrxPI) in E. coli and a fraction was biotinylated. A double paratope model was used in which samples were incubated with TrxPI-biotin and microspheres adsorbed with TrxPI. The immune complexes were revealed using Streptavidin-Phycoerythrin. The geometric mean of the signals was analyzed, and the results were expressed as Standard Deviation scores (SDs). Sera from 100 normal human control and from 111 type 1 diabetic patients were evaluated by FloCMIA. To correlate the novel assay with RBA, 51 diabetic patients were selected, spanning a wide range of PAA reactivity by RBA. Results: The study of ROC curves allowed choosing a cut-off value of 3.0 SDs and the AUC was 0.705, indicating that FloCMIA has fair ability to distinguish between samples from each group. A prevalence of 50% for PAA was obtained in the population of diabetic patients studied. The specificity was 96% and the analytical sensitivity (percentage of patients RBA positive, also positive by FloCMIA) was 69%. There was a substantial agreement between methods (kappa statistic=0.700). Conclusions: A novel immunoassay based on flow cytometry that uses easy-to produce recombinant PI was developed. This assay constitutes an innovative and cost-effective alternative to RBA for the determination of PAA in patients' sera. The method developed here, presents good performance and a wide dynamic range together with a small required sample volume. Furthermore, these results make it possible to develop multiplex immunoassays that allow the combined detection of autoantibodies present in T1DM and other related autoimmune diseases.

Biogenesis of the Insulin Secretory Granule in Health and Disease.

The secretory granules of pancreatic beta cells are specialized organelles responsible for the packaging, storage and secretion of the vital hormone insulin. The insulin secretory granules also contain more than 100 other proteins including the proteases involved in proinsulin-to insulin conversion, other precursor proteins, minor co-secreted peptides, membrane proteins involved in cell trafficking and ion translocation proteins essential for regulation of the intragranular environment. The synthesis, transport and packaging of these proteins into nascent granules must be carried out in a co-ordinated manner to ensure correct functioning of the granule. The process is regulated by many circulating nutrients such as glucose and can change under different physiological states. This chapter discusses the various processes involved in insulin granule biogenesis with a focus on the granule composition in health and disease.

Islet-Derived CD4 T Cells Targeting Proinsulin in Human Autoimmune Diabetes.

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing ß-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.

Reduced ß-Cell Secretory Capacity in Pancreatic-Insufficient, but Not Pancreatic-Sufficient, Cystic Fibrosis Despite Normal Glucose Tolerance.

Patients with pancreatic-insufficient cystic fibrosis (PI-CF) are at increased risk for developing diabetes. We determined ß-cell secretory capacity and insulin secretory rates from glucose-potentiated arginine and mixed-meal tolerance tests (MMTTs), respectively, in pancreatic-sufficient cystic fibrosis (PS-CF), PI-CF, and normal control subjects, all with normal glucose tolerance, in order to identify early pathophysiologic defects. Acute islet cell secretory responses were determined under fasting, 230 mg/dL, and 340 mg/dL hyperglycemia clamp conditions. PI-CF subjects had lower acute insulin, C-peptide, and glucagon responses compared with PS-CF and normal control subjects, indicating reduced ß-cell secretory capacity and α-cell function. Fasting proinsulin-to-C-peptide and proinsulin secretory ratios during glucose potentiation were higher in PI-CF, suggesting impaired proinsulin processing. In the first 30 min of the MMTT, insulin secretion was lower in PI-CF compared with PS-CF and normal control subjects, and glucagon-like peptide 1 and gastric inhibitory polypeptide were lower compared with PS-CF, and after 180 min, glucose was higher in PI-CF compared with normal control subjects. These findings indicate that despite "normal" glucose tolerance, adolescents and adults with PI-CF have impairments in functional islet mass and associated early-phase insulin secretion, which with decreased incretin responses likely leads to the early development of postprandial hyperglycemia in CF.

[Endogenous hyperinsulinism: two diagnostic challenges]. / Hiperinsulinismo endógeno: dois desafios diagnósticos.

Hypoglycemia in apparently healthy adults is a rare finding in clinical practice requiring a thorough investigation of the cause. During the investigation, identification of hypoglycemia associated with inappropriately high levels of insulin and C-peptide should prompt the exclusion of rare causes of hypoglycemia, including pancreatic islet-cells disease and autoimmune hypoglycemia. In this paper, we describe two cases of hypoglycemia associated with endogenous hyperinsulinism, whose causes are uncommon in clinical practice, and review important aspects of the diagnosis and treatment of hyperinsulinemic hypoglycemia.

Hiperinsulinismo endógeno: dois desafios diagnósticos / Endogenous hyperinsulinism: two diagnostic challenges

A hipoglicemia em um adulto aparentemente saudável é um achado raro na prática clínica que exige uma investigação exaustiva da causa. A identificação de glicemia plasmática diminuída associada a concentrações plasmáticas de insulina e peptídeo-C não suprimidos deverá levar à exclusão de causas raras de hipoglicemia, entre elas, doença das células betapancreáticas e hipoglicemia autoimune. Neste artigo, descrevemos dois casos de hipoglicemia associada a hiperinsulinismo endógeno, cujas causas são pouco habituais na prática clínica. A propósito desses casos clínicos revemos aspectos importantes de diagnósticos e tratamento da hipoglicemia no contexto de hiperinsulinismo endógeno.

Hypoglycemia in apparently healthy adults is a rare finding in clinical practice requiring a thorough investigation of the cause. During the investigation, identification of hypoglycemia associated with inappropriately high levels of insulin and C-peptide should prompt the exclusion of rare causes of hypoglycemia, including pancreatic islet-cells disease and autoimmune hypoglycemia. In this paper, we describe two cases of hypoglycemia associated with endogenous hyperinsulinism, whose causes are uncommon in clinical practice, and review important aspects of the diagnosis and treatment of hyperinsulinemic hypoglycemia.

Triiodothyronine (T3) induces proinsulin gene expression by activating PI3K: possible roles for GSK-3ß and the transcriptional factor PDX-1.

Thyroid hormone (TH) activates PI3K and Akt, leading to glucose uptake in rat skeletal muscle cells and proliferation of insulinoma cells, respectively. However, TH actions on pancreatic beta cells have been little explored, which lead us to evaluate the TH eff ects on proinsulin gene expression, and the involvement of PI3K/Akt/GSK-3ß signaling pathway, and a transcriptional factor for insulin (PDX-1). INS-1E cells were sorted into 3 groups: control and TH-depleted treated or not with T3 for 30 min. Cells were also previously treated with actinomycin D (ActD), cycloheximide (CHX), wortmannin or Akt inhibitor. Proinsulin mRNA expression was evaluated by real time PCR, and pGSK-3ß and PDX-1 protein content was analyzed by Western blotting. TH depletion decreased proinsulin mRNA content, which was restored after acute T3 treatment. ActD, CHX and wortmannin, but not Akt inhibitor, prevented the rapid stimulatory eff ect of T3 on proinsulin mRNA expression. TH depletion did not affect the phosphorylated GSK-3ß and PDX-1 protein content; but T3 treatment led to an increase in the content of these proteins. These data indicate that T3 acutely increases proinsulin mRNA expression, by mechanisms which depends on the activation of PI3K, but not of Akt, and may involve the inactivation of GSK-3ß by phosphorylation. Since GSK-3ß enhances PDX-1 degradation rate, the GSK-3ß inactivation could explain the increase of PDX-1 content in T3-treated cells. Considering that PDX-1 is one of the most important transcriptional factors for proinsulin gene expression, its enhancement may underlie the increased proinsulin mRNA content acutely induced by T3.

Surface plasmon resonance reveals a different pattern of proinsulin autoantibodies concentration and affinity in diabetic patients.

Type 1 diabetes mellitus (DM) is characterized by autoimmune aggression against pancreatic beta cells resulting in absolute deficiency of insulin secretion. The first detectable sign of emerging autoimmunity during the preclinical asymptomatic period is the appearance of diabetes-related autoantibodies. In children at risk for type 1 DM, high-affinity Insulin autoantibodies reactive to proinsulin, are associated with diabetes risk. Autoantibodies are usually measured by radioligand binding assay (RBA) that provides quasi-quantitative values reflecting potency (product between concentration and affinity) of specific autoantibodies. Aiming to improve the characterization of the specific humoral immune response, we selected surface plasmon resonance (SPR) as an alternative method to measure proinsulin autoantibodies (PAA). This novel technology has allowed real time detection of antibodies interaction and kinetic analysis. Herein, we have employed SPR to characterize the PAA present in sera from 28 childhood-onset (mean age 8.31±4.20) and 23 adult-onset diabetic patients (≥65 years old, BMI<30) in terms of concentration and affinity. When evaluating comparatively samples from both groups, childhood-onset diabetic patients presented lower PAA concentrations and higher affinities (median 67.12×10(-9) M and 3.50×10(7) M(-1), respectively) than the adults (median 167.4×10(-9) M and 0.84×10(7) M(-1), respectively). These results are consistent with those from the reference method RBA (Standard Deviation score median 9.49 for childhood-onset group and 5.04 for adult-onset group) where the binding can be directly related to the intrinsic affinity of the antibody, suggesting that there is a different etiopathogenic pathway between both types of clinical presentation of the disease. This technology has shown to be a useful tool for the characterization of PAAs parameters as an alternative to radioimmunoassay, with high versatility and reproducibility associated to low occupational and environmental risk. However, this technology is not eligible for routine marker screening, but this is a powerful technique for a fine description of the thermodynamic parameters of antigen-antibody interaction.

Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli.

Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its high proteolytic decay and troubles to reproduce the native disulphide pattern. In the present study, human PI was produced in E. coli as a fusion thioredoxin protein (Trx-PI). Such chimeric protein was obtained from the intracellular soluble fraction, and it was purified in one step by affinity chromatography on immobilized phenylarsine oxide. Trx-PI was also recovered from inclusion bodies and purified by anion exchange chromatography. The product identity and integrity were verified by mass analysis (22,173.5 Da) and mapping with Staphylococcus aureus V8 protease. Native PI folding was evaluated by biochemical and also by immunochemical analysis using specific sera from PI antibody-positive diabetic patients that recognise conformational discontinue epitopes. Dose-response curves showed identity between standard PI and Trx-PI. Moreover, surface plasmon resonance technique verified the correct conformation of the recombinant protein. The biochemical and immunochemical assays demonstrated the integrity of the chimera and the epitopes involved in the interaction with antibodies. In conclusion, it was possible to obtain with high-yield purified human PI as a fusion protein in E. coli and useful for analytical purposes.

Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris.

Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris.

Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells.

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Alterations of NADPH oxidase activity in rat pancreatic islets induced by a high-fat diet.

OBJECTIVE: The aim of this study was to evaluate the effect of a high-fat diet (HFD) on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in rat pancreatic islets. We investigated if changes in NADPH oxidase are connected to beta cell dysfunction reported in obese animals. METHODS: Male Wistar rats were fed a HFD or control diet for 3 months. DNA fragmentation, insulin secretion, and [U-C]glucose oxidation were examined in isolated pancreatic islets. The oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal were assessed by immunohistochemistry. The protein content of gp91 and p47 was evaluated by Western blotting. Production of reactive oxygen species (ROS) was determined by a fluorescence assay using hydroethidine. RESULTS: Occurrence of DNA fragmentation was reduced in pancreatic islets from HFD rats. There were no differences in oxidative stress markers between the groups. Glucose oxidation and insulin secretion were elevated due to high glucose in pancreatic islets from HFD rats. Protein concentrations of p47 and gp91 subunits were reduced and ROS production was diminished in pancreatic islets from HFD rats. CONCLUSIONS: The diminished content of NADPH oxidase subunits and ROS concentrations may be associated with increased glucose oxidation and insulin secretion in an attempt to compensate for the peripheral insulin resistance elicited by the HFD.

NAD(P)H oxidase participates in the palmitate-induced superoxide production and insulin secretion by rat pancreatic islets.

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose-stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free-albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI--diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase-I (CPT-I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47(PHOX) translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up-regulated the protein content of p47(PHOX) and the mRNA levels of p22(PHOX), gp91(PHOX), p47(PHOX), proinsulin and the G protein-coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate.

Serum levels of adiponectin and leptin in children born small for gestational age: relation to insulin sensitivity parameters.

Children born small for gestational age (SGA) are prone to developing obesity, insulin resistance and type 2 diabetes. Adiponectin and leptin are adipocytokines associated with insulin sensitivity parameters. We aimed to relate serum adiponectin and leptin levels with insulin sensitivity parameters in prepuberal SGA children with and without catch-up growth (SGA+CUG; SGA-CUG, respectively) and to analyze the usefulness of these adipocytokines as early markers of insulin resistance. We analysed adiponectin, proinsulin, leptin, growth factors, insulin, HOMA IR and HOMA beta(cell) in 23 SGA+CUG, 26 SGA-CUG children compared with 48 prepuberal appropiate for gestational age (AGA). SGA children had adiponectin levels comparable to AGA children. Leptin levels were different between sexes, showed to be higher in SGA+CUG group (p=0.040) and these were significantly correlated with insulin sensitivity parameters. These results suggest leptin resistance as an adaptive mechanism to increase energy balance, but an altered functional response of adipocytes cannot be discarded.

Correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds.

Soybean plants are promising bioreactors for the expression of biochemically complex proteins that cannot be produced in a safe and/or economically viable way in microorganisms, eukaryotic culture cells or secreted by transgenic animal glands. Soybeans present many desirable agronomic characteristics for high scale protein production, such as high productivity, short reproductive cycle, photoperiod sensitivity, and natural organs destined for protein accumulation in the seeds. The significant similarities between plant and human cells in terms of protein synthesis processes, folding, assembly, and post-translational processing are important for efficient accumulation of recombinant proteins. We obtained two transgenic lines using biolystics, incorporating the human proinsulin gene under control of the monocot tissue-specific promoter from sorghum gamma-kafirin seed storage protein gene and the alpha-coixin cotyledonary vacuolar signal peptide from Coix lacryma-jobi (Poaceae). Transgenic plants expressed the proinsulin gene and accumulated the polypeptide in mature seeds. Protein targeting to cotyledonary protein storage vacuoles was successfully achieved and confirmed with immunocytochemistry assays. The combination of different regulatory sequences was apparently responsible for high stability in protein accumulation, since human proinsulin was detected after seven years under room temperature storage conditions.

Beta-cell adenomas without hyperinsulinemia with use of highly specific insulin radioimmunoassays: case report and review of literature.

OBJECTIVE: To report a case of a proinsulin-secreting islet cell adenoma in which the diagnosis was obscured by an ultraspecific insulin assay. METHODS: We describe the case of a 46-year-old woman, who presented with fasting hypoglycemia and appropriately low insulin values. RESULTS: A prolonged supervised fast produced symptomatic hypoglycemia (20 mg/dL) after only 7 hours. During the entire fasting test, highly specific insulin remained at <3 mIU/L, with a median value (and interquartile range) of 0.9 (0.8 to 2.3) mIU/L, when the glucose concentration was <50 mg/dL. The serum C-peptide level remained high normal (mean +/- SD, 2.7 +/- 0.6 ng/mL; normal fasting levels, 0.8 to 3.9), and no evidence of sulfonylurea use was detected in the patient's urine. Circulating proinsulin levels were persistently high (>200 pmol/L in all determinations when hypoglycemia was present; expected value, <5 pmol/L). Magnetic resonance imaging and endoscopic ultrasonography confirmed the presence of a 2.5-cm tumor in the head of the pancreas. A proinsulin-secreting islet cell tumor was diagnosed. Surgical resection of the tumor was successfully accomplished, but diabetes mellitus developed 4 months postoperatively. CONCLUSION: The diagnosis of a hypoglycemia-producing pancreatic adenoma can be missed when an ultraspecific insulin assay is used. The direct measurement of proinsulin established the diagnosis in this case.