Biochemical and Biophysical Research Communications
Evidence for the expression of both the hydrolase and translocase components of hepatic glucose-6-phosphatase in murine pancreatic islets
Section snippets
Methods
Tissue isolation and RNA extraction. Pancreata were removed from ob/ob mice, chopped, and digested for 10–12 min with continuous shaking (150 strokes/min) at 37 °C in 2 ml of Hanks’ balanced salt solution containing 6 mg collagenase. Islets were collected under stereomicroscopy and employed immediately for RNA extraction. The β cell line MIN6 was maintained in DMEM (glucose 11 mM) supplemented with 15% fetal calf serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin sulfate, and 50 μM β-mercaptoethanol,
Expression of hepatic G6Pase catalytic subunit in pancreatic islets
In order to demonstrate the expression of the catalytic subunit of hepatic G6Pase in pancreatic islets, RT-PCR was performed with cDNA prepared from ob/ob islet total RNA. With appropriate optimization and use of a sufficient quantity of cDNA (see below), PCR products were obtained when using primers amplifying between nucleotides 168–649, 295–658, 577–865, 577–961, and 1713–1972 (see Figs. 1A–E, respectively) of the mouse hepatic G6Pase catalytic subunit sequence. In each case, the product
Discussion
In view of the fact that G6Pase activity has been measured in mouse pancreatic islets [13], [22], [23], the expression of components of the G6Pase system was studied in ob/ob mouse islets. Using primer pairs targeting various regions of the G6Pase mRNA, it was possible to demonstrate the amplification from ob/ob mouse islet cDNA of PCR products with sizes identical to those obtained from liver cDNA. In addition, using a PCR-based approach in which a region of the entire open reading frame was
Acknowledgements
This work was supported by the Swedish Medical Research Council (Grant K2003/72BI/14624/01A), Novo-Nordisk, and Karo Bio AB.
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