Proinsulin Research Articles

Proinsulin and the proinsulin/insulin ratio in overweight and obese children and adolescents: relation to clinical parameters, insulin resistance, and impaired glucose regulation

In adults with impaired glucose regulation (IGR) or type 2 diabetes mellitus (T2DM), proinsulin (PI) and its ratio to insulin (ins; PI/I ratio) are frequently elevated. Here we assessed the relationship among fasting PI, clinical parameters, and carbohydrate metabolism, a potential difference of the PI/I ratio between overweight and obese youth with or without IGR in an oral glucose tolerance test (OGTT) and the predictive power of PI levels for IGR. In a cross-sectional study, data of n = 259 children and adolescents attending our obesity clinic were studied. Fasting PI levels were determined in all patients and matched to the standard assessment of obesity. In n = 154 subjects at risk for T2DM, an OGTT was performed sampling for glucose, ins, and PI. ins, glucose, PI, PI/I ratio, insulin resistance [homeostasis model assessment of insulin resistance index (HOMA-IR)]. Puberty (by Tanner) and relative body weight [body mass index (BMI)-standard deviation of BMI (SDS)] showed a linear relationship to fasting PI levels (p < 0.001 for Tanner I vs. II-V; p = 0.04 and p = 0.026 for BMI-SDS < 2 vs. 2-2.5 and 2-2.5 vs. > 2.5, respectively). Subjects with ins resistance (HOMA-IR > 95th percentile, n = 140) had higher fasting PI levels than those without (p < 0.001), with no significant difference in fasting PI/I ratio (p > 0.05). As compared to subjects with normal glucose regulation, subjects with IGR (n = 35) had higher fasting PI (p = 0.001) and an increased PI/I ratio, both during fasting and at 30 min during OGTT (p = 0.049 and p = 0.014, respectively). Children and adolescents with IGR have disproportionately elevated PI levels, both during fasting and after acute glucose stimulation indicating β-cell dysfunction.

An investigation of the relationship of inflammatory response and insulin and its components during stress hyperglycemia in critically ill patients

To observe the relationship between inflammatory response and the constituents of islet β cell secretion during stress hyperglycemia (SHG) in critically ill patients, in order to study the impact of inflammatory response on insulin resistance and the secretion function of islet β cells. According to the state of inflammatory response, 45 critical patients with SHG were divided into two groups: stress and the convalescence period. Twenty five healthy individuals were enrolled as control group. The blood levels of tumour necrosis factor β (TNF-β), blood glucose (BG), and insulin components including proinsulin (PI), immunoreactive insulin (IRI), true insulin (TI), C-peptide (C-P) were measured respectively. The levels of BG, TNF-β, insulin components, insulin resistance index (HOMA-IR) and the secretion index (HOMA-β) were compared among groups. The relationship between TNF-β and BG, insulin components, HOMA-IR, HOMA-β were analyzed. (1)There was no difference in concentrations of TI among stress period, convalescence stage and control group [3.68 (1.57, 7.70), 3.42 (2.41, 7.40), 1.46 (0.35, 4.90) mU/L, all P >0.05], whereas the concentration of BG [(10.04 ± 2.43) mmol/L], TNF-β [13.70 (11.77, 20.00) ng/L], PI [6.20 (3.22, 9.27) pmol/L], IRI [13.45 (9.88, 19.88) mU/L] and C-P [3.01 (2.37, 4.00) μg/L]in stress period were significantly higher than those in the convalescence stage[BG: (6.09 ± 0.84) mmol/L, TNF-β: 11.58 (8.80, 13.22) ng/L,PI: 1.54 (0.36, 11.82) pmol/L, IRI: 10.80 (5.35, 12.60) mU/L, C-P: 2.42 (1.17, 3.56) μg/L] and control group [BG: (4.87 ± 0.56) mmol/L,TNF-β: 9.27 (7.48, 12.16) ng/L, PI: 2.20 (1.88, 4.54) pmol/L, IRI: 5.50 (4.00, 8.00) mU/L, C-P: 1.15 (0.87, 1.76) μg/L, P <0.05 or P <0.01]. (2)The HOMA-IR [5.17 (3.41, 11.51)] in stress period was significantly higher than that in the convalescence[3.24 (1.51, 6.95)] and control group [1.14 (0.81, 1.79), P <0.05 and P<0.01]. The HOMA-β [10.80 (3.72, 31.40)] of isletβ cell in stress period was significantly lower than that in the convalescence [28.42 (6.46, 125.01)] and control group [21.94 (7.77, 62.01), P <0.01 and P <0.05]. (3)There were positive correlations between the concentration of TNF-β and PI, IRI, C-P and HOMA-IR ( r 1=0.292, r 2=0.344, r 3=0.397, r 4=0.324, P <0.05 or P <0.01). There were negative correlation between concentration of TNF-β and HOMA-β ( r =-0.235 , P <0.05) . The severer the inflammatory response, the higher PI, IRI and C-P, while the secretion of TI is relatively deficient.Inflammatory response could affect insulin resistance and the secretion function of islet βcell during SHG in critically ill patients.

Intramuscular delivery of a naked DNA plasmid encoding proinsulin and pancreatic regenerating III protein ameliorates type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by inflammation of pancreatic islets and destruction of β cells. Up to now, there is still no cure for this devastating disease and alternative approach should be developed. To explore a novel gene therapy strategy combining immunotherapy and β cell regeneration, we constructed a non-viral plasmid encoding proinsulin (PI) and pancreatic regenerating (Reg) III protein (pReg/PI). Therapeutic potentials of this plasmid for T1DM were investigated. Intramuscular delivery of pReg/PI resulted in a significant reduction in hyperglycemia and diabetes incidence, with an increased insulin contents in the serum of T1DM mice model induced by STZ. Treatment with pReg/PI also restored the balance of Th1/Th2 cytokines and expanded CD4 +CD25 +Foxp3 + T regulatory cells, which may attribute to the establishment of self-immune tolerance. Additionally, in comparison to the mice treated with empty vector pBudCE4.1 (pBud), attenuated insulitis and apoptosis achieved by inhibiting activation of NF-κB in the pancreas of pReg/PI treated mice were observed. In summary, these results indicate that intramuscular delivery of pReg/PI distinctly ameliorated STZ-induced T1DM by reconstructing the immunological self-tolerance and promoting the regeneration of β cells, which might be served as a promising candidate for the gene therapy of T1DM.

Relationship between adiponectin and the first phase of glucose-stimulated insulin secretion from pancreatic β-cell in patients with type 2 diabetes mellitus

Objective To investigate the relationship between adiponectin and the first-phase of pancreatic P-cell insulin secretion in subjects with different statuses of glucose tolerance. Methods Thirty-seven patients with newly diagnosed type 2 diabetes mellitus (DM) , 30 patients with abnormal glucose tolerance (IGR) , and 40 normal control subjects (NGT) underwent intravenous glucose tolerance test (IVGTT). Fasting adiponectin and proinsulin (PI) was assayed by EL1SA. Fasting free fatty acid ( FFA) was measured by colorimetry. Insulin area under the curve ( AUC ) , incremental AUC (iAUC) from 0 min to 10 min, AIR3-5, homeostasis model assessment for insnlin resistance (HOMA-IR) , and for β cell function ( HOMA-p) were calculated. The relationship between adiponectin and AUC, iAUC, AIR3-5, proinsulin, FFA, and HOMA-IR was explored. Results (1) The levels of AUC, iAUC, AIR3-5, and adiponectin in DM group and IGR group were significantly lower than those in NGT group (P<0.05), reduced in DM group than those in IGR group(P<0.05). (2) The levels of PI in DM group and IGR group were significantly higher than that in NGT (P<0.05). (3) Adiponectin was positively correlated with HOMA-p,AUC,iAUC,AIR3-5, and HDL-C,while negatively correlated with proinsulin, HOMA-IR, and LDL-C. (4) Proinsulin was positively correlated with HOMA-IR. (5 ) Multiple regression stepwise analysis showed that adiponectin was independently associated with AUC. Conclusions Adiponectin was an independent factor affecting the first phase of pancreatic p-cell insulin secretion. Low adiponectin level could predict the dysfunction of the first phase pancreatic p-cell secretion as well as insulin resistance in patients with type 2 diabetes. Key words: Adiponectin; Intravenous glucose tolerance test; First-phase of insulin secretion; Proinsulin

Insulin gene VNTR genotype associates with frequency and phenotype of the autoimmune response to proinsulin

Immune responses to autoantigens are in part controlled by deletion of autoreactive cells through genetically regulated selection mechanisms. We have directly analyzed peripheral CD4+ proinsulin (PI) 76–90 (SLQPLALEGSLQKRG)-specific T cells using soluble fluorescent major histocompatibility complex class II tetramers. Subjects with type I diabetes and healthy controls with high levels of peripheral proinsulin-specific T cells were characterized by the presence of a disease-susceptible polymorphism in the insulin variable number of tandem repeats (INS-VNTR) gene. Conversely, subjects with a ‘protective' polymorphism in the INS-VNTR gene had nearly undetectable levels of proinsulin tetramer-positive T cells. These results strongly imply a direct relationship between genetic control of autoantigen expression and peripheral autoreactivity, in which proinsulin genotype restricts the quantity and quality of the potential T-cell response. Using a modified tetramer to isolate low-avidity proinsulin-specific T cells from subjects with the susceptible genotype, transcript arrays identified several induced pro-apoptotic genes in the control, but not diabetic subjects, likely representing a second peripheral mechanism for maintenance of tolerance to self antigens.

Establishment of electrochemiluminescence immunoassay to detect proinsulin levels in human serum

Objective To establish a electrochemilumineecenee immunoaasay (ECLIA) for detecting proinsulin(PI) levels in human serum. Methods PI ECLIA kit contains biotinylated monoclonal insulin-specificantibody, ruthenium labeled monoclonal C-peptide specific antibody, and streptavidin-coated microparticlea. Its method performance, such as sensitivity, specific, accuracy, was evaluated. Then, the levels of PI in clinical samples were detected by ECLIA, including 44 type 2 diabetes and 60 healthy volunteers. Results Average recovery of ECLIA was 103. 8% with a lower detection limit of 0.38 pmol/L and biologic detection limit between 0.25 pmol/L and 0.5 pmol/L, and the function sensitivity was 0.5 pmol/L. The human insulin and human C-peptide did not cross-react at 500 μU/ml recovery of ECLIA was 103.8% with a lower detection limit of 0.38 pmol/L and biologic detection limit between 0.25 pmol/L and 0.5 pmol/L, and the function sensitivity was 0.5 pmoL/L. The human insulin and human C-peptide did not cross-react at 500 μU/ml and 450 ng/ml respectively. The total coefficient of variation of the ECLIA were leas than 5.0%, and the analytical measure range (AMR) was between 2.22 and 169.68 pmol/L. There was a good correlation (r=0.967,P=0.000) between ECLIA and radio immunoassay (RIA). The 95% reference value for human proinsulin was 1.07-15.54 pmol/L. The plasma levels of PI in type 2 diabetes were significantly higher than those in control healthy group[6.72(3.61-11.92) pmol/L vs 5.72(2.65-8.74) pmol/L, Z=-2.023, P<0.05]. Conclusions The monoclonal-based ECLIA is a sensitive, specific, and rapid method and no radiocontamination. It can be used to detect hanum serum proinsulin in type 2 diabetes. Key words: Proinsulin; Electrochemistry; Luminescence; Immunoassay

Utility of Early Insulin Response and Proinsulin to Assess Insulin Resistance

To determine whether obesity and premature adrenarche are additive events increasing the risk of insulin resistance and beta-cell failure, using early insulin response (EIR) or the insulinogenic index and proinsulin (PI) as markers. This was a prospective case-control study conducted at a tertiary care academic medical center involving 81 prepubertal, predominantly Hispanic children (34 males, 47 females), classified as lean controls (4 males, 6 females; mean age, 6.5 +/- 1.2 years; mean body mass index [BMI] z-score, 0.08 +/- 0.6), obese controls (20 males, 10 females; mean age, 7.2 +/- 1.5 years; mean BMI z-score, 2.5 +/- 0.5), lean premature adrenarche (3 males, 11 females; mean age, 7.1 +/- 1.2 years; mean BMI z-score, 0.09 +/- 0.6), and obese premature adrenarche (7 males, 20 females; mean age, 7.3 +/- 1.0; mean BMI z-score, 2.2 +/- 0.4). Fasting glucose (G(0)), insulin (I(0)), PI(0), androgen levels, insulin-like growth factor 1, insulin-like growth factor binding protein 1, and lipid levels were obtained. An oral glucose tolerance test was performed. EIR was calculated as (I(30) - I(0))/(G(30) - G(0)). Between-group differences were assessed by 2-way analysis of variance, with interactions and associations explored with correlation/regression. EIR was greater in the obese subjects with and without premature adrenarche. Combined analysis of the independent variables obesity and premature adrenarche showed that the obese premature adrenarche group had the highest EIR. The obese subjects with premature adrenarche had greater fasting PI levels than their lean counterparts. The differences in fasting PI/I ratio among the groups were not statistically significant. Using EIR and PI as markers to assess the risk of insulin resistance and impaired insulin secretion indicates that obese children with premature adrenarche may be at greater risk for the development of prediabetes and type 2 diabetes mellitus compared with their lean counterparts.

Maintenance of Pancreatic Endocrine Cells of Neonatal Rats: Effect of 2-Deoxyglucose on Insulin Biosynthesis

The relationship between insulin biosynthesis and proinsulin mRNA activity was examined in pancreatic monolayer cultures of the neonatal rat. Monolayer cultures were maintained in TCM 199 medium containing 16.7 mM of either glucose or 2-deoxyglucose or in a basal medium not containing glucose for 24 hr. The fractions containing mRNA extracted from these cultures were translated in a cell-free protein synthesizing system of rabbit reticulocyte lysate. The proinsulin mRNA activity was determined by the radioactivity of the translation product immunoprecipitated using anti-insulin serum, and identified as preproinsulin with a molecular weight of 11,500 on gel electrophoresis. The amount of insulin biosynthesis was determined by the incorporation of [3H]-leucine into (pro)insulin. In islet B cells cultured in a medium with 16.7 mM glucose, insulin biosynthesis induced by 16.7 mM glucose was enhanced by 158% when cultured in basal medium without glucose. This increment corresponded nicely with a 185% increase in the proinsulin mRNA activity. However, the addition of 16.7 mM 2-deoxyglucose to the basal medium resulted in a 293% increase in glucose-induced insulin biosynthesis despite a 16% drop in the proinsulin mRNA activity, and primed a dose-dependent increase of insulin biosynthesis over the concentration range of 0 to 16.7 mM glucose. These results suggest first that in neonatal B cells insulin biosynthesis may be regulated at the transcriptional level, and second, that 2-deoxyglucose may cause the transition of the neonatal B cell to an adult-type response.

In Vitro and in Vivo Behaviour of Rat Islets of Langerhans Treated with MHC Antisera and Complement2)

The in vitro and in vivo behaviour of rat islets of Langerhans pretreated with polyclonal MHC antisera and complement was investigated. Complement-dependent cytotoxicity against rat islets was mediated by the antisera which were either directed against all MHC products ("a anti-u") or against products of the B-region of rat MHC ("Ba anti-Bu"). There were only minor alterations in glucose-stimulated insulin secretion and in 3H-leucine incorporation into islet proteins and into (pro)insulin in the pretreated islets. The syngeneic transplantation of "Ba anti-Bu"-treated islets led to a permanent graft survival in all streptozotocin-diabetic rats but after "a anti-u"-pretreatment permanent graft survival was only achieved in 33% of the syngeneic recipients. It is concluded, that in the latter case a complement-dependent cytotoxicity is exerted against the islets which may not allow sufficient in vivo survival. Now it appears necessary to look for effects of pretreatment prior to an allogeneic transplantation.

Effect of serum from diabetes-prone BB/OK rats on neonatal rat pancreatic islets and islet cell suspensions.

Humoral-mediated cytolytic activity against neonatal rat pancreatic islet cells as well as islets of Langerhans was detected in sera of newly diagnosed diabetic BB/OK rats by measurement of enhanced 51Cr-release or insulin leakage. Beta-cell-specific functions of islet cell suspensions such as insulin secretion and (pro)insulin biosynthesis can be affected by BB rat serum in vitro.

Proinsulin in pregnant women with normal glucose tolerance or mild gestational diabetes mellitus.

Pregnancy is characterized by peripheral insulin resistance, which is physiologically compensated by an increase in insulin secretion. Type 2 diabetes and impaired glucose tolerance (IGT) have been associated with an inappropriate increase in insulin precursors, namely proinsulin. The aim of this study was to determine levels of proinsulin (PI), specific insulin (SI) and the proinsulin-to-specific insulin (PI/SI) ratio in consecutive pregnant women (n = 209) with normal glucose tolerance (NGT), as assessed by a 2h oral glucose tolerance test, and with mild gestational diabetes (GDM), in comparison to 32 healthy, non-pregnant women. Furthermore, we related these variables to surrogate markers of insulin resistance and insulin secretion. We found no significant differences in the levels of PI and the PI/ SI ratio between pregnant and non-pregnant women (PI: 5.0 +/- 3.6 vs. 4.8 +/- 3.5 pmol/L, p = NS), and between pregnant women with mild GDM and NGT (PI: 5.4 +/- 2.4 vs. 4.9 +/- 3.9 pmol/L, p = NS). SI was elevated in women with mild GDM (112.2 +/- 47.3 vs. 94.8 +/- 43.0 pmol/L in NGT, p=0.02). PI was related to fasting glucose (r = 0.17, p < 0.02), but not post-load glucose levels, and to fasting insulin [specific insulin: r = 0.67, p = 0.0001; total immunoreactive insulin (IRI): r = 0.69, p = 0.0001], as well as post-load insulin levels (IRI at 120 min: r = 0.18, p < 0.03). The PI/SI ratio showed no association with fasting or post-load glucose or insulin levels. Pregnant women presented with a metabolic pattern suggestive of enhanced insulin resistance, namely increased fasting and post-load insulin levels. In women with mild GDM, fasting and post-load hyperglycemia, as well as an additional increase in insulin resistance was found. Group differences weakened when accounting for differences in body weight. The data of the present study suggest that in normal pregnancy as well as mild GDM metabolic alterations including enhanced insulin resistance and hyperglycemia do not result in an increase in circulating levels of proinsulin, both in absolute terms and relative to levels of specific insulin, as indicated by the proinsulin-to-specific insulin ratio.

Proinsulin gene therapy in diabetic rats——Comparison of the effects on blood glucose by intraportal infusion and intramuscular injection

Objective To compare the effects of rat proinsulin gene therapy via intraportal infusion and intramuscular injection blood glucose level in streptozotocin-induced diabetic rots. Methods (1) Recombinant eukaryotic cell expression plasmid of rat proinsulin gene pCMV/proiusulin was transferred into streptozotocin-induced diabetic rats by intraportal infusion and intramuscular injection to observe the effect of rat proiusulin gene therapy in diabetic rats. The treatment group by intraportal infusion (group A) and the group by intramuscular injection (group C) were given pCMV/proinsulin naked plasmid DNA 100 μg, while the control groups by intraportal infusion (group B) or by intramuscular injection (group D) were treated with similar amount of pCMV DNA. Normal group and diabetes mellitus group were also observed at the same time. (2) Blood glucose level was tested and serum insulin was determined by radioimmunoassay. RT-PCR and immunohistochemistry were used to detemine proinsulin mRNA and protein expressions in liver and skeletal muscle and protein. Results (1) The blood glucose levels in two treated groups were both decreased. In group A, levels of blood sugar decreased about 7 mmol/L and glycemie control was maintained for 3-4 weeks. Serum insulin levels step up significantly after pCMV/proinsulin gene therapy. The blood glucose level in group A was significantly lower than those of group B and DM group (P<0.05), while the serum insulin level was higher than those of two groups (P<0.05). In group C, blood glucose levels decreased about 4 mmol/L and glycemic control was maintained for 1-2 weeks. Meanwhile, the concentrations of insulin increased markedly after gene therapy. The blood glucose in group C was significantly lower than those of group D and DM group (P<0.05), while the serum insulin level was higher than those of two groups (P<0.05). (2) Proinsulin mRNA and protein expressions could be detected in either hepatic cell of group A or skeletal muscle cell of group C, not in group B and group D. Conclusion Proiusulin genetherapy via intraportal infusion or intramuscular injection lowers significantly blood glucose in diabetic rats, and thus offers a potential approach to treatment of diabetes. Key words: Diabetes mellitus; Gene therapy; Proinsulin gene; Intraportal infusion; Intramuscular injection

Unexplained severe hypoglycaemia in hospital: a difficult diagnostic challenge

Unexplained severe hypoglycaemia in hospital: a difficult diagnostic challenge

Dipeptidyl-Peptidase-IV Inhibition Augments Postprandial Lipid Mobilization and Oxidation in Type 2 Diabetic Patients

Abstract Context: Dipeptidyl-peptidase-IV (DPP-4) inhibition increases endogenous GLP-1 activity resulting in improved glycemic control in patients with type 2 diabetes mellitus. The metabolic response may be explained in part by extra-pancreatic mechanisms. Objective: We tested the hypothesis that DPP-4 inhibition with vildagliptin elicits changes in adipose tissue and skeletal muscle metabolism. Design: Randomized, double blind, crossover study. Setting: Academic clinical research center. Patients: Twenty patients with type 2 diabetes, body mass index between 28 and 40 kg/m2. Intervention: Seven days treatment with the selective DPP-4 inhibitor vildagliptin or placebo. Standardized test meal on day seven. Main Outcome Measures: Venous DPP-4 activity, catecholamines, free fatty acids, glycerol, glucose, (pro)insulin; dialysate glucose, lactate, pyruvate, glycerol. Results: Fasting and postprandial venous insulin, glucose, glycerol, triglycerides and free fatty acid concentrations were not different with vildagliptin and with placebo. Vildagliptin augmented the postprandial increase in plasma norepinephrine. Furthermore, vildagliptine increased dialysate glycerol and lactate concentrations in adipose tissue while suppressing dialysate lactate and pyruvate concentration in skeletal muscle. The respiratory quotient increased with meal ingestion but was consistently lower with vildagliptin. Conclusions: Our study is the first to suggest that DPP-4 inhibition augments postprandial lipid mobilization and oxidation. The response may be explained by sympathetic activation rather than a direct effect on metabolic status.

Total error profiling of a proinsulin time-resolved fluorescence immunoassay

We applied total error profiling to evaluate the conversion of a known proinsulin (PI) enzyme-linked immunosorbent assay (ELISA) into a time-resolved fluorescence immunoassay (TRFIA). The formula and acceptance criteria proposed by the Ligand Binding Assay Bioanalytical Focus Group (LBABFG) of the American Association of Pharmaceutical Scientists (AAPS) were applied. We found that the expected dynamic range enlargement with TRFIA compared to ELISA ([0.5–240] versus resp. [0.7–98] pmol/L) is limited by an interference of C-peptide when present in the sample at high concentrations (>7000 pmol/L).

Mutated ATP synthase induces oxidative stress and impaired insulin secretion in β‐cells of female BHE/cdb rats

Adenosine triphosphate (ATP) is a critical determinant of beta-cell insulin secretion in response to glucose. BHE/cdb rats have a mutation in ATP synthase that limits ATP production, yet develop mild diabetes only with ageing. We investigated the cellular basis for reduced insulin secretion and compensatory mechanisms that mitigate the effects of the ATP synthase mutation. In vitro beta-cell function in isolated islets and expression of key regulatory genes was compared with in vivo oral glucose tolerance and insulin sensitivity in BHE/cdb and control rats. BHE/cdb rat islets had reduced responsiveness to glucose stimulation and ATP content was 35% lower than in control islets. Oral glucose tolerance was impaired at both 21 and 43 weeks of age because of a reduction in glucose-stimulated insulin secretion (GSIS). An increase in inducible nitric oxide synthase (INOS, 3-fold) and manganese superoxide dismutase (MnSOD, 1.6-fold), detection of nitrotyrosine, beta-cell apoptosis, and nucleocytoplasmic translocation of pancreas duodenum homeobox-1 (PDX-1) in beta-cells indicated increased oxygen radical formation. However, BHE/cdb rats partially compensated for low glucose responsiveness by increasing the number of small islets and beta-cell hypertrophy. There was also an increase in the proportion of mature insulin relative to proinsulin (PI) detected within beta-cell granules. Increased activation of AMP-dependent kinase (AMPK)-regulated pathways was consistent with increased oxidative stress and with induction of apoptosis and reduction of preproinsulin gene transcription. The findings are consistent with impaired but partially compensated mechanisms of insulin secretion early in life, but progressive non-compensated impairments due to oxidative stress occurs by age 43 weeks.

Reduced postprandial proinsulinaemia and 32–33 split proinsulinaemia after a mixed meal in type 2 diabetic patients following sensitization to insulin with pioglitazone

Reduced insulin sensitivity associated with fasting hyperproinsulinaemia is common in type 2 diabetes. Proinsulinaemia is an established independent cardiovascular risk factor. The objective was to investigate fasting and postprandial release of insulin, proinsulin (PI) and 32-33 split proinsulin (SPI) before and after sensitization to insulin with pioglitazone compared to a group treated with glibenclamide. A randomized double-blind placebo-controlled trial. Twenty-two type 2 diabetic patients were recruited along with 10 normal subjects. After 4 weeks washout, patients received a mixed meal and were assigned to receive pioglitazone or glibenclamide for 20 weeks, after which patients received another identical test meal. The treatment regimes were designed to maintain glycaemic control (HbA1c) at pretreatment levels so that beta-cells received an equivalent glycaemic stimulus for both test meals. Plasma insulin, PI, SPI and glucose concentrations were measured over an 8-h postprandial period. The output of PI and SPI was measured as the integrated postprandial response (area under the curve, AUC). Pioglitazone treatment resulted in a significant reduction in fasting levels of PI and SPI compared to those of the controls. Postprandially, pioglitazone treatment had no effect on the insulin AUC response to the meal but significantly reduced the PI and SPI AUCs. Glibenclamide increased fasting insulin and the postprandial insulin AUC but had no effect on the PI and SPI AUCs. Sensitization to insulin with pioglitazone reduces the amount of insulin precursor species present in fasting and postprandially and may reduce cardiovascular risk.

Proinsulin maturation, misfolding, and proteotoxicity

As a tool to explore proinsulin (PI) trafficking, a human PI cDNA has been constructed with GFP fused within the C peptide. In regulated secretory cells containing appropriate prohormone convertases, the hProCpepGFP construct undergoes endoproteolytic processing to CpepGFP and native human insulin, which are specifically detected and cosecreted in parallel with endogenous insulin. Expression of C(A7)Y mutant PI results in autosomal dominant diabetes in Akita mice. We directly identify the misfolded PI in Akita islets and also show that C(A7)Y mutant PI, either in the context of the hProCpepGFP chimera or not, engages directly in protein complexes with nonmutant PI, impairing the trafficking and recovery of nonmutant PI. This trapping mechanism decreases insulin production in beta cells. Thereafter we observe a loss of beta cell viability. The data imply that PI misfolding leading to impaired endoplasmic reticulum exit of nonmutant PI may be a key early step in a chain reaction of beta cell dysfunction and demise leading to onset and progression of diabetes.

A MUFA-Rich Diet Improves Posprandial Glucose, Lipid and GLP-1 Responses in Insulin-Resistant Subjects

Objective: To study the effects of three weight-maintenance diets with different macronutrient composition on carbohydrate, lipid metabolism, insulin and incretin levels in insulin-resistant subjects.Methods: A prospective study was performed in eleven (7 W, 4 M) offspring of obese and type 2 diabetes patients. Subjects had a BMI > 25 Kg/m2, waist circumference (men/women) > 102/88, HBA1c < 6.5% and were regarded as insulin-resistant after an OGTT (Matsuda ISIm <4). They were randomly divided into three groups and underwent three dietary periods each of 28 days in a crossover design: a) diet high in saturated fat (SAT), b) diet rich in monounsaturated fat (MUFA; Mediterranean diet) and c) diet rich in carbohydrate (CHO).Results: Body weight and resting energy expenditure did not changed during the three dietary periods. Fasting serum glucose concentrations fell during MUFA-rich and CHO-rich diets compared with high-SAT diets (5.02 ± 0.1, 5.03 ± 0.1, 5.50 ± 0.2 mmol/L, respectively. Anova < 0.05). The MUFA-rich diet improved insulin sensitivity, as indicated by lower homeostasis model analysis-insulin resistance (HOMA-ir), compared with CHO-rich and high-SAT diets (2.32 ± 0.3, 2.52 ± 0.4, 2.72 ± 0.4, respectively, Anova < 0.01). After a MUFA-rich and high-SAT breakfasts (443 kcal) the postprandial integrated area under curve (AUC) of glucose and insulin were lowered compared with isocaloric CHO-rich breakfast (7.8 ± 1.3, 5.84 ± 1.2, 11.9 ± 2.7 mmol · 180 min/L, Anova < 0.05; and 1004 ± 147, 1253 ± 140, 2667 ± 329 pmol · 180 min/L, Anova <0.01, respectively); while the integrated glucagon-like peptide-1 response increased with MUFA and SAT breakfasts compared with isocaloric CHO-rich meals (4.22 ± 0.7, 4.34 ± 1.1, 1.85 ± 1.1, respectively, Anova < 0.05). Fasting and postprandial HDL cholesterol concentrations rose with MUFA-rich diets, and the AUCs of triacylglycerol fell with the CHO-rich diet. Similarly fasting proinsulin (PI) concentration fell, while stimulated ratio PI/I was not changed by MUFA-rich diet.Conclusions: Weight maintenance with a MUFA-rich diet improves HOMA-ir and fasting proinsulin levels in insulin-resistant subjects. Ingestion of a virgin olive oil-based breakfast decreased postprandial glucose and insulin concentrations, and increased HDL-C and GLP-1 concentrations as compared with CHO-rich diet.

HLA Class I Epitope Discovery in Type 1 Diabetes

Type 1 diabetes mellitus (T1DM) results from the destruction of beta cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.