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

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*

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

Animals ; Autoantigens ; genetics ; metabolism ; Cell Line, Tumor ; Golgi Apparatus ; genetics ; metabolism ; Golgi Matrix Proteins ; Insulin-Secreting Cells ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Proinsulin ; genetics ; metabolism ; Rats ; rab1 GTP-Binding Proteins ; genetics ; metabolism

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

OBJECTIVE: To evaluate the effect of hepatic insulin gene therapy on diabetic enteric neuropathy. METHODS: Mice were randomly allocated into 3 groups: a normal control group, a diabetic group, and a diabetic gene therapy group. Diabetes were induced by penial vein injection of streptozocin (STZ). The gene therapy group received hepatic insulin gene therapy while the other 2 groups only received an empty virus expressing green fluorescent protein. Random blood glucose, body weight growth, gastric emptying, total bowel length, absolute and relative bowel transit, electric field stimulation of colon smooth muscle, colon nuclei staining and counting were measured. RESULTS: We successully established a mouse model of diabetic enteric neuropathy which manifests as: 8 weeks of continuous hyperglycemia,increased total bowel length, decreased relative bowel transit, impaired colon smooth muscle relaxation and loss of inhibitory neurons in colon. Through gene therapy, the above indexes were normalized or ameliorated, suggesting hepatic insulin gene therapy is capable of preventing diabetic enteric neuropathy. CONCLUSION Hepatic insulin gene therapy can prevent STZ induced diabetic enteric neuropathy.
Adenoviridae ; Animals ; Diabetes Mellitus, Experimental ; complications ; therapy ; Diabetic Neuropathies ; therapy ; Enteric Nervous System ; metabolism ; pathology ; Gastrointestinal Diseases ; etiology ; therapy ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Hepatocytes ; metabolism ; Insulin ; genetics ; metabolism ; Mice ; Proinsulin ; genetics

The aim of this study is to investigate the therapeutic effect of RegIII-proinsulin-pBudCE4.1 plasmid on streptozotocin (STZ)-induced type 1 diabetes mellitus and its underlying mechanisms. The model of type 1 diabetes mellitus was established by intraperitoneal injections of STZ (40 mg kg(-1)) to Balb/c mice for five consecutive days. Then, ten type 1 diabetic mice were intramuscularly injected with 100 microg RegIII-proinsulin-pBudCE4.1 plasmid for 4 weeks (one time/week) and the blood glucose levels were monitored every week; whereas another ten diabetic mice served as negative control group were injected with pBudCE4.1 vector at the same dose. Normal control and model control mice were treated with normal saline at identical volume under the same way. Western blotting, MTT assay, ELISA, HE staining and Tunel assay were applied to explore the underlying mechanisms. Results showed that RegIII-proinsulin-pBudCE4.1 plasmid ameliorated the hyperglycemia symptoms in diabetic mouse remarkably. It induced an immunological tolerance state in type 1 diabetic mice by inhibiting the proliferation of splenic lymphocytes and recovering Th1/Th2 balance evidenced by MTT and ELISA analysis. Furthermore, it elevated insulin concentration in the serum of type 1 diabetic mice and promoted the regeneration of beta cells supported by the results of HE staining and Tunel assay. In conclusion, RegIII-proinsulin-pBudCE4.1 plasmid possesses powerful anti-diabetic ability, which may be involved in the inducing of immunological tolerance and enhancing beta cells recovery.
Animals ; Apoptosis ; Blood Glucose ; metabolism ; Cell Proliferation ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; therapy ; Diabetes Mellitus, Type 1 ; chemically induced ; metabolism ; pathology ; therapy ; Genetic Therapy ; Hyperglycemia ; therapy ; Injections, Intramuscular ; Insulin ; blood ; Islets of Langerhans ; cytology ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; Proinsulin ; genetics ; metabolism ; therapeutic use ; Proteins ; genetics ; metabolism ; therapeutic use ; Streptozocin ; T-Lymphocytes ; cytology ; Th1-Th2 Balance

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: 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

OBJECTIVETo investigate the difference between serum true insulin (TI) and immunoreactive insulin (IRI) in evaluating islet beta-cell function and insulin resistance.

METHODSThe arginine stimulation test was performed in 141 individuals, including 35 with normal glucose tolerance (NGT) and 106 with type 2 diabetes (T2DM). Plasma glucose (PG), TI, IRI and proinsulin (PI) levels were measured; the incremental value of TI/PG, (TI+PI)/PG and IRI/PG (delta TI/PG, delta(TI+PI)/PG and deltaIRI/PG) and the area under curve of TI/PG, (TI+PI)/PG and IRI/PG (AUC [TI/PG], AUC[(TI+PI)/PG] and AUC [IRI/PG]) after arginine stimulation were calculated to evaluate beta-cell function.

RESULTThere were positive correlations of delta TI/PG with delta (TI+PI)/PG and delta IRI/PG in both NGT and T2DM patients (r=0.68 - 0.99, P<0.01). The similar correlations of AUC [TI/PG] with AUC [(TI+PI)/PG] and AUC [IRI/PG] were also shown (r=0.62 - 0.99, P<0.01). delta TI/PG was correlated with AUC [TI/PG] in two groups (NGT r=0.96, T2DM r=0.82, P<0.01). HOMA-IRTI, HOMA-IR(TI+PI) and HOMA-IRIRI in T2DM were higher than those in NGT (P<0.01). After arginine stimulation T2DM subjects mainly presented insulin resistance and decreased insulin secretion.

CONCLUSIONThe determination of TI may be more accurate than IRI in evaluating beta-cell function and insulin resistance.

Adult ; Aged ; Arginine ; pharmacology ; Blood Glucose ; metabolism ; Diabetes Mellitus, Type 2 ; blood ; Female ; Glucose Tolerance Test ; Humans ; Insulin ; blood ; immunology ; Insulin Resistance ; Insulin-Secreting Cells ; physiology ; Male ; Middle Aged ; Proinsulin ; blood