Evidence for the expression of both the hydrolase and translocase components of hepatic glucose-6-phosphatase in murine pancreatic islets

https://doi.org/10.1016/S0006-291X(03)01242-7Get rights and content

Abstract

Glucose-6-phosphatase (G6Pase) is a multicomponent enzyme system which regulates the catalysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6Pase can antagonize glucose phosphorylation, a step prerequisite in the regulation of insulin secretion from pancreatic β cells, and G6Pase activity is increased in islets isolated from animal models of type II diabetes. Using RT-PCR with hepatic G6Pase catalytic subunit primers, we demonstrate that the sizes of amplified products from ob/ob mouse islets are identical to those from liver cDNA. This was confirmed by PCR-based cloning and sequencing of the hepatic G6Pase catalytic subunit open reading frame from islet cDNA. The expression in islets of the G6P transporter, G6PT1, was also demonstrated, suggesting that all of the identified hepatic G6Pase system genes are expressed in pancreatic islets. Finally, the expression of islet-specific G6Pase-related protein (IGRP) in pancreatic islets was confirmed and its expression in liver was also observed.

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.

References (31)

  • B Lin et al.

    Cloning and characterization of cDNAs encoding a candidate glycogen storage disease type 1b protein in rodents

    J. Biol. Chem.

    (1998)
  • A Khan et al.

    Effects of 3-mercaptopicolinic acid and a derivative of chlorogenic acid (S-3483) on hepatic and islet glucose-6-phosphatase activity

    Eur. J. Pharmacol.

    (1998)
  • C.C Martin et al.

    Cloning and characterization of the human and rat islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) genes

    J. Biol. Chem.

    (2001)
  • R.C Nordlie
  • G van der Werve et al.

    New lessons in the regulation of glucose metabolism taught by the glucose-6-phosphatase system

    Eur. J. Biochem.

    (2000)
  • Cited by (4)

    • Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

      2013, Biochimica et Biophysica Acta - General Subjects
      Citation Excerpt :

      It has been observed for a long time that rodent pancreatic islets possess G6Pase activity [27–29]. Rodent ß-cells also express the G6PC/G6PT mRNA [30]. Both the enzyme activity and the G6PC protein appear to become expressed – together with the glucose-induced insulin secretion – in the INS1 rat insulinoma cell line as compared to the parent, glucose-insensitive, RINm5F cells [31].

    • Anaplerotic roles of pyruvate carboxylase in mammalian tissues

      2006, Cellular and Molecular Life Sciences
    View full text