Pulsatile insulin release from islets isolated from three subjects with type 2 diabetes.

Plasma insulin in healthy subjects shows regular oscillations, which are important for the hypoglycemic action of the hormone. In individuals with type 2 diabetes, these regular variations are altered, which has been implicated in the development of insulin resistance and hyperglycemia. The origin of the change is unknown, but derangement of the islet secretory pattern has been suggested as a contributing cause. In the present study, we show the dynamics of insulin release from individually perifused islets isolated from three subjects with type 2 diabetes. Insulin release at 3 mmol/l glucose was 10.5 +/- 4.5 pmol.g(-1).s(-1) and pulsatile (0.26 +/- 0.05 min(-1)). In islets from one subject, 11 mmol/l glucose transiently increased insulin release by augmentation of the insulin pulses without affecting the frequency. Addition of 1 mmol/l tolbutamide did not increase insulin release. In islets from the remaining subjects, insulin release was not affected by 11 mmol/l glucose. Tolbutamide transiently increased insulin release in islets from one subject. Insulin release from four normal subjects at 3 mmol/l glucose was 4.3 +/- 0.8 pmol.g(-1).s(-1) and pulsatile (0.23 +/- 0.03 min(-1)). At 11 mmol/l glucose, insulin release increased in islets from all subjects. Tolbutamide further increased insulin release in islets from two subjects. It is concluded that islets from the three individuals with type 2 diabetes release insulin in pulses. The impaired secretory response to glucose may be related to impaired metabolism before mitochondrial degradation of the sugar.

Site-directed mutations in the FAD-binding domain of glycerophosphate dehydrogenase: catalytic defects with preserved mitochondrial anchoring of the enzyme in transfected COS-7 cells.

Single-strand conformational polymorphism analysis of mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH) gene has revealed mutations in both the calcium- and FAD-binding domains of this enzyme in some diabetic patients. It was now investigated whether site-directed mutations in the FAD-binding domain of the mGDH gene may affect the mitochondrial anchoring and catalytic activity of the enzyme. COS-7 cells were transfected with plasmid cDNA coding for either wild-type or mutated human mGDH (G --> A substitutions at positions 352, 355, and 364 and A --> C substitution at position 390) fused, when required, at the N-terminus of green fluorescent protein. The activity of mGDH was measured by both radioisotopic ((3)HOH production from l-[2-(3)H]glycerol 3-phosphate) and colorimetric (iodoformazan formation) procedures. In cells transfected with the mGDHwt-EGFP or mGDHmut-EGFP constructs, the fused protein was found by confocal microscopy exclusively in the mitochondria, colocalized with a mitochondrial marker. In homogenates of COS-7 cells transfected with mGDHmut, however, the catalytic activity of the enzyme was decreased, this coinciding with low ratios between both the activities measured in the absence/presence of exogenous FAD and the results obtained by the colorimetric/radioisotopic procedure. Thus, although the present site-directed mutations of the mGDH gene failed to impair the mitochondrial anchoring of the enzyme, they led to catalytic defects that were, in some respect, comparable to those previously encountered in the lymphocytes or islets of type 2 diabetic patients.