Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells.

AIMS/HYPOTHESIS: Of the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4's role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells. METHODS: Endogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)-RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes. RESULTS: Syn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs. CONCLUSIONS/INTERPRETATION: Syn-4 is involved in distinct molecular machineries that influence exocytosis of both pre-docked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4's role in mediating portions of first-phase and second-phase GSIS.

Modeling of 24-hour glucose and insulin profiles in patients with type 2 diabetes mellitus treated with biphasic insulin aspart.

Insulin therapy for diabetes patients is designed to mimic the endogenous insulin response of healthy subjects and thereby generate normal blood glucose levels. In order to control the blood glucose in insulin-treated diabetes patients, it is important to be able to predict the effect of exogenous insulin on blood glucose. A pharmacokinetic/pharmacodynamic model for glucose homoeostasis describing the effect of exogenous insulin would facilitate such prediction. Thus the aim of this work was to extend the previously developed integrated glucose-insulin (IGI) model to predict 24-hour glucose profiles for patients with Type 2 diabetes following exogenous insulin administration. Clinical data from two trials were included in the analysis. In both trials, 24-hour meal tolerance tests were used as the experimental setup, where exogenous insulin (biphasic insulin aspart) was administered in relation to meals. The IGI model was successfully extended to include the effect of exogenous insulin. Circadian variations in glucose homeostasis were assessed on relevant parameters, and a significant improvement was achieved by including a circadian rhythm on the endogenous glucose production in the model. The extended model is a useful tool for clinical trial simulation and for elucidating the effect profile of new insulin products.

Decreased active GLP-1 response following large test meal in patients with type 1 diabetes using bolus insulin analogues.

Postprandial plasma immunoreactive active glucagon-like peptide-1 (p-active GLP-1) levels in type 1 diabetic patients who did not use bolus insulin responded normally following ingestion of test meal, while a small response of p-active GLP-1 levels was seen in type 2 diabetic patients. To determine whether p-active GLP-1 levels are affected by ingestion of test meal in type 1 diabetic Japanese patients who used bolus rapid-acting insulin analogues, plasma glucose (PG), serum immunoreactive insulin (s-IRI), serum immunoreactive C-peptide (s-CPR), and p-active GLP-1 levels were measured 0, 30, and 60 min after ingestion of test meal in Japanese patients without diabetic complications (n=10, group 1) and control subjects with normal glucose tolerance (n=15, group 2). HbA1c levels were also measured in these groups. The patients in group 1 were treated with multiple daily injections or CSII using injections of bolus rapid-acting insulin analogues before ingestion of test meal. There was no significant difference in mean of sex, age, or BMI between groups. Means of HbA1c, basal and postprandial PG, and postprandial s-IRI levels with integrated areas under curves (0-60 min) (AUC) in group 1 were significantly higher than those in group 2. Means of basal and postprandial s-CPR, and postprandial p-active GLP-1 levels with AUCs were significantly lower in group 1 than in group 2. These results indicated that postprandial p-active GLP-1 levels following ingestion of test meal in type 1 diabetic Japanese patients using bolus rapid-acting insulin analogues were decreased relative to those in controls.