Background: Insulin is a hormone produced by the beta \r\n', u'cells of the pancreas that permits glucose to enter cells and \r\n', u'helps the body use glucose for energy. Insulin controls the \r\n', u'amount of glucose in the blood. Insulin is produced by recombinant protein technology. Expression of human proinsulin is the first step to express insulin. Objectives:To express successfully human proinsulin gene in pET 28a vector and E.coli BL21 (DE3). Subjects and method: Human proinsulin gene was applied from human pencreas cDNA by PCR using specific PINS primer pairs which contained sites for BamH I, Xho I. Proinsulin gene was cloned into pET 28a (+) vector to form recombinant vector pET 28a-PINS then transformed into E.coli BL21 (DE3) host strain to make pET 28a-PINS/ BL21 (DE3) clone. The clone was cultured and induced by IPTG (1mM). Recombine protein was analysed by SDS-PAGE. Results: Expression vector pET 28a-PINS was constructed successfully. Proinsulin protein expressed in E.coli BL21 (DE3) was purified by ProPond-Resin (Amersham). Conclusion: Human proinsulin was produced successfully using pET 28a-PINS/ BL21 (DE3) system.\r\n', u'
Proinsulin/ pharmacology ; pharmacokinetics ; Escherichia coli

Introduction: There are thousands of diabetes sufferers worldwide. In addition, there is an increased trend in Vietnam due to economic development, increased population, lifespan, and changing lifestyle. Insulin is a hormone, which is a natural protein. It plays an important role in the transformation of glucose in human and animal blood. Note, while insulin has not been produced in Vietnam, the production of recombinant Proinsulin is a premise for insulin. This study is based on a successful design of pET - 28a (+) Vector with recombinant Proinsulin codified gene.\r\n', u'Objectives: To define the human proinsulin codified gene and study the optimum conditions for the expression of proinsulin. \r\n', u'Subjects and method: To discover the appropriate conditions for the expression of proinsulin, including initial cell density, cultural temperature, IPTG concentration, and expression time. The product of the expression was confirmed by Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SOS - PAGE) and reconfirmed by Western blotting with His - Tag antibody. \r\n', u'Results: Proinsulin expression was successfully proved by SOS - PAGE and Western blotting. Four appropriate conditions for the expression were confirmed as highlighted in the Conclusion. \r\n', u'Conclusion: The appropriate conditions for expression of proinsulin: cell density was 00600 0.6 - 1.0; the cultural temperature was 300C; IPTG concentration was 0.4 mM; the length of the culture time was 20 hours. \r\n', u'
Expression ; Proinsulin ; E.Coli ; Appropriate conditions

BACKGROUND: Although insulin resistance and decreased insulin secretion are characteristics of establised type 2 DM, which of these metabolic abnormalities is the primary determinant of type 2 DM is controversial. It is also not well known how insulin resistance and beta cell dysfunction influence serum insulin, proinsulin, proinsulin/insulin ratio in type 2 DM. METHODS: We compared serum insulin, proinsulin, proinsulin/insulin ratio in type 2 diabetic patients and control subjects. We investigated the relationship between serum insulin, proinsulin, proinsuolin/insulin ratio and several biochemical markers which represent insulin resistance or beta cell function. RESULTS: Proinsulin, total insulin, and proinsulin/insulin ratio were significantly higher in type 2 DM than control(p< 0.001). In diabetic patients, proinsulin was correlated with fasting c-peptide(r=0.43, p=0.002), postprandial 2 hour blood glucose(r=0.213, p=0.05), and triglyceride(r=0.28, p=0.022). Total insulin was correlated with urinary albumin excretion rates(r=0.224, p=0.025) and body mass index(r=0.269, p=0.014). Proinsulin/insulin ratio was positively correlated with fasting c-peptide(r=0.236, p=0.031), fasting blood glucose(r=0.264, p=0.015), postprandial 2 hour blood glucose(r=0.277, p=0.001), and triglyceride(r=0.428, p< 0.001). In control subjects, fasting insulin was correlated with triglyceride (r=0.366, p=0.002). Proinsulin/insulin ratio was correlated with age(r=0.241, p=0.044). CONCLUSION: The serum levels of insulin and proinsulin seems to be associated with several markers of insluin resistance. The proinsulin/insulin ratio might represent beta cell function rather than insulin resistance. But more studies are needed to clarify the mechanism of elevated proinsulin/insulin ratio in type 2 DM.
Biomarkers ; Fasting ; Humans ; Insulin Resistance ; Insulin* ; Proinsulin* ; Triglycerides

BACKGROUND: Measurement of insulin and C-peptide concentrations is important for deciding whether insulin treatment is required in diabetic patients. We aimed to investigate the analytical performance of insulin and C-peptide assays using the Lumipulse G1200 system (Fujirebio Inc., Tokyo, Japan). METHODS: We examined the precision, linearity, and cross-reactivity of insulin and C-peptide using five insulin analogues and purified proinsulin. A method comparison was conducted between the Lumipulse G1200 and Roche E170 (Roche Diagnostics, Mannheim, Germany) systems in 200 diabetic patients on insulin treatment. Reference intervals for insulin and C-peptide concentrations were determined in 279 healthy individuals. RESULTS: For insulin and C-peptide assays, within-laboratory precision (% CV) was 3.78–4.14 and 2.89–3.35%, respectively. The linearity of the insulin assay in the range of 0–2,778 pmol/L was R2=0.9997, and that of the C-peptide assay in the range of 0–10 nmol/L was R2=0.9996. The correlation coefficient (r) between the Roche E170 and Lumipulse G1200 results was 0.943 (P < 0.001) for insulin and 0.996 (P < 0.001) for C-peptide. The mean differences in insulin and C-peptide between Lumipulse G1200 and the Roche E170 were 19.4 pmol/L and 0.2 nmol/L, respectively. None of the insulin analogues or proinsulin showed significant cross-reactivity with the Lumipulse G1200. Reference intervals of insulin and C-peptide were 7.64–70.14 pmol/L and 0.17–0.85 nmol/L, respectively. CONCLUSIONS: Insulin and C-peptide tests on the Lumipulse G1200 show adequate analytical performance and are expected to be acceptable for use in clinical areas.
C-Peptide* ; Diabetes Mellitus ; Humans ; Insulin* ; Methods ; Proinsulin

More effective production of human insulin is important, because insulin is the main medication that is used to treat multiple types of diabetes and because many people are suffering from diabetes. The current system of insulin production is based on recombinant DNA technology, and the expression vector is composed of a preproinsulin sequence that is a fused form of an artificial leader peptide and the native proinsulin. It has been reported that the sequence of the leader peptide affects the production of insulin. To analyze how the leader peptide affects the maturation of insulin structurally, we adapted several in silico simulations using 13 artificial proinsulin sequences. Three-dimensional structures of models were predicted and compared. Although their sequences had few differences, the predicted structures were somewhat different. The structures were refined by molecular dynamics simulation, and the energy of each model was estimated. Then, protein-protein docking between the models and trypsin was carried out to compare how efficiently the protease could access the cleavage sites of the proinsulin models. The results showed some concordance with experimental results that have been reported; so, we expect our analysis will be used to predict the optimized sequence of artificial proinsulin for more effective production.
Computer Simulation ; DNA, Recombinant ; Humans* ; Insulin ; Molecular Dynamics Simulation* ; Proinsulin ; Protein Sorting Signals ; Trypsin

BACKGROUND AND OBJECTIVES: In advanced β-cell dysfunction, proinsulin is increasingly replacing insulin as major component of the secretion product. It has been speculated that proinsulin has at least the same adipogenic potency than insulin, leading to an increased tendency of lipid tissue formation in patients with late stage β-cell dysfunction. METHODS AND RESULTS: Mesenchymal stem cells obtained from liposuction material were grown in differentiation media containing insulin (0.01 μmol), proinsulin (0.01 μmol) or insulin+proinsulin (each 0.005 μmol). Cell culture supernatants were taken from these experiments and an untreated control at weeks 1, 2, and 3, and were stored at −80°C until analysis. Cell differentiation was microscopically supervised and adiponectin concentrations were measured as marker for differentiation into mature lipid cells. This experiment was repeated three times. No growth of lipid cells and no change in adiponectin values was observed in the negative control group (after 7/14/12 days: 3.2±0.5/3.3±0.1/4.4±0.5 ng/ml/12 h). A continuous differentiation into mature adipocytes (also confirmed by Red-Oil-staining) and a corresponding increase in adiponectin values was observed in the experiments with insulin (3.6±1.9/5.1±1.4/13.3±1.5 ng/ml/12 h; p < 0.05 week 1 vs. week 3) and proinsulin (3.3±1.2/3.5±0.3/12.2±1.2 ng/ml/12 h; p < 0.05). Comparable effects were seen with the insulin/proinsulin combination. CONCLUSIONS: Proinsulin has the same adipogenic potential than insulin in vitro. Proinsulin has only 10~20% of the glucose-lowering effect of insulin. It can be speculated that the adipogenic potential of proinsulin may be a large contributor to the increased body weight problems in patients with type 2 diabetes and advanced β-cell dysfunction.
Adipocytes* ; Adiponectin ; Body Weight ; Cell Culture Techniques ; Cell Differentiation ; Humans ; In Vitro Techniques* ; Insulin* ; Lipectomy ; Mesenchymal Stromal Cells* ; Proinsulin*

Insulinoma with hyperproinsulinemia and normal serum insulin level is a rare disease. Because of the neuroglycopenic symptoms, the initial diagnosis tends to be made as epilepsy or as psychosis. A 43-year-old man was admitted to our hospital because of recurrent confusional episodes. Symptoms are intermittent and consist of staring, confusion, amnesia, and bizarre behavior. Vital signs during the episode were normal but the serum glucose level was 27 mg/dl. The serum level of insulin during the episode was lower than normal and those of proinsulin and growth hormone were higher than normal. Solitary pancreatic mass was found by abdominal CT, measuring 15 mm in diameter. Pathologic evaluation showed islet cell tumor. This suggests that the serum level of proinsulin should be checked when insulinoma with neuroglycopenic symptom is suspected.
Adenoma, Islet Cell ; Adult ; Amnesia ; Blood Glucose ; Diagnosis ; Epilepsy ; Growth Hormone ; Humans ; Insulin ; Insulinoma* ; Proinsulin ; Psychotic Disorders ; Rare Diseases ; Seizures* ; Tomography, X-Ray Computed ; Vital Signs

The sensitive and specific immunoradiometric assay is described for human proinsulin and its intermediate peptides (65-66 split and 32-33 split proinsulin). We developed a monoclonal antibody-based two-site immunoradiometric assay with use of streptavidin-biotin labeling. The detection limits of the assays lie in the range of 0.5-2.0 pM. In the proinsulin assay proinsulin cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. In the assay of 65-66 split proinsulin it does not cross-react with insulin, proinsulin or 32-33 split proinsulin. In the 32-33 split proinsulin assay it cross-reacted 84% with proinsulin and 60% with 65-66 split proinsulin. The precision (C.V.) of the assays was less than 15% over the various concentration. The mean concentrations of insulin, proinsulin, 65-66 split proinsulin and 32-33 proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 3.6, 2.3 +/- 0.3, undetectable (less than 1.0) and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4, 3.8 +/- 0.6 and 19.7 +/- 6.0 respectively.
Adult ; Animals ; Antibodies, Monoclonal/*immunology ; Bacterial Proteins ; Biotin ; Cross Reactions ; Humans ; Immunoradiometric Assay ; Male ; Mice ; Proinsulin/*analysis/immunology ; Reproducibility of Results ; Streptavidin

BACKGROUND: Multiple laboratory tests are used in the diagnosis and management of patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially. APPROACH: An expert committee compiled evidencebased recommendations for the use of laboratory analysis in patients with diabetes. A new system was developed to grade the overall quality of the evidence and the strength of the recommendations. A draft of the guidelines was posted on the Internet, and the document was modified in response to comments. The guidelines were reviewed by the joint Evidence-Based Laboratory Medicine Committee of the AACC and the National Academy of Clinical Biochemistry and were accepted after revisions by the Professional Practice Committee and subsequent approval by the Executive Committee of the American Diabetes Association. CONTENT: In addition to the long-standing criteria based on measurement of venous plasma glucose, diabetes can be diagnosed by demonstrating increased hemoglobin A1c (HbA1c) concentrations in the blood. Monitoring of glycemic control is performed by the patients measuring their own plasma or blood glucose with meters and by laboratory analysis of Hb A1c. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of autoantibodies, urine albumin, insulin, proinsulin, C-peptide, and other analytes are addressed. SUMMARY: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are found to have minimal clinical value at the present time, and measurement of them is not recommended.
Autoantibodies ; Biochemistry ; Blood Glucose ; C-Peptide ; Consensus ; Diabetes Mellitus ; Genetic Testing ; Glucose ; Hemoglobin A, Glycosylated ; Hemoglobins ; Humans ; Insulin ; Internet ; Joints ; Plasma ; Professional Practice ; Proinsulin

OBJECTIVEThe strong relation between type 2 diabetes mellitus and obesity with acanthosis nigricans is widely concerned. This study investigated the pancreatic beta-cell function in obese children with acanthosis nigricans, so as to find out the role of insulin secretion and insulin resistance in obese children with acanthosis nigricans.

METHODSThirty-five obese children with acanthosis nigricans (19 males and 16 females with mean age 12.8 +/- 1.5 years) were enrolled in this study. Thirty-eight obese children (21 boys and 17 girls with mean age 11.9 +/- 2.6 years) and 39 normal children (20 boys and 19 girls with mean age 11.2 +/- 2.2 years) were recruited as obese and normal control groups. The levels of serum fasting insulin, C-peptide, proinsulin and true insulin were measured in all the subjects. The ratios of proinsulin/insulin and proinsulin/C-peptide were calculated. Homeostasis model assessment was applied to assess the status of insulin resistance and basic function of pancreatic beta-cell.

RESULTSThe levels of fasting insulin, C-peptide proinsulin, true insulin, the ratios of proinsulin/insulin and proinsulin/C-peptide, insulin resistance index and insulin secretion index of obese children with acanthosis nigricans, obese control children and normal control children were: 18.5 (5.0-60.5) pmol/L, 12.4 (6.1-35.8) pmol/L and 5.1 (2.0-32.8) pmol/L; 3.9 (1.3-14.0) microg/L, 2.4 (1.1-4.0) microg/L and 1.1 (1.0-4.2) microg/L; 28.8 (9.9-64.2) pmol/L, 9.5 (2.2-34.5) pmol/L and 4.2 (2.0-16.0) pmol/L; 33.0 (6.2-66.0) pmol/L, 10.6 (4.8-29.4) pmol/L and 4.5 (1.3-30.1) pmol/L; 1.2 (0.4-8.9), 0.9 (0.2-1.9) and 0.8 (0.4-2.0); 6.9 (2.5-36.6), 4.7 (1.2-12.3) and 3.6 (1.2-9.6); 5.0 (0.8-14.1), 2.6 (1.3-8.1) and 1.2(0.4-6.9); 303.3 (52.2-1,163.8), 213.6 (84.6-572.0) and 51.1 (19.1-561.4). The levels of fasting insulin, C-peptide, proinsulin, true insulin, the ratios of proinsulin/insulin and proinsulin/C-peptide, insulin resistance index and insulin secretion index in obese children with acanthosis nigricans were significantly higher than those in obese children (P < 0.001) and normal children (P < 0.001).

CONCLUSIONObese children with acanthosis nigricans had higher insulin resistance and pancreatic beta-cell dysfunction; acanthosis nigricans may be a skin sign of high risk of type 2 diabetes mellitus.

Acanthosis Nigricans ; complications ; Adolescent ; C-Peptide ; blood ; Child ; Diabetes Mellitus, Type 2 ; etiology ; Female ; Humans ; Insulin ; blood ; Insulin Resistance ; Islets of Langerhans ; physiopathology ; Male ; Obesity ; complications ; physiopathology ; Proinsulin ; blood