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

BACKGROUND: Although insulin resistance and decreased insulin secretion are characteristics of established 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 and proinsulin/insulin ratio in type 2 diabetic patients and control subjects. We also investigated the relationship between serum insulin, proinsulin and proinsulin/insulin ratio and several biochemical markers which represent insulin resistance or beta cell function. RESULTS: Insulin, proinsulin and proinsulin/insulin ratio were significantly higher in type 2 DM than control(p < 0.001). In diabetic patients, total insulin level was correlated with urinary albumin excretion rates(r = 0.224, p = 0.025) and body mass index(r = 0.269, p = 0.014). Proinsulin level 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). 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, insulin level 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 seem to be associated with several markers of insulin resistance. Whereas proinsulin/insulin ratio might represent beta cell function rather than insulin resistance. But more studies are needed to clarify the mechanisms of elevated proinsulin/insulin ratio in type 2 DM.
Aged ; Diabetes Mellitus, Non-Insulin-Dependent/etiology ; Diabetes Mellitus, Non-Insulin-Dependent/blood* ; Female ; Human ; Insulin/blood* ; Insulin Resistance* ; Islets of Langerhans/physiopathology* ; Male ; Middle Age ; Proinsulin/blood* ; Sulfonylurea Compounds/pharmacology

OBJECTIVETo construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expression in myotube cell line.

METHODSAn expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recombined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentrations of 0, 2, and 10 microg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits.

RESULTSImmune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day.

CONCLUSIONHuman insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.

Animals ; Cell Line ; Dose-Response Relationship, Drug ; Doxycycline ; pharmacology ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; genetics ; Insulin ; biosynthesis ; genetics ; Mice ; Muscle Fibers, Skeletal ; cytology ; metabolism ; Proinsulin ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

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