Fuel and hormone regulation of phospholipase C β1 and δ1 overexpressed in RINm5F pancreatic beta cells
Introduction
Insulin secretion from the pancreatic beta cell is dependent on the coordinated and temporal inputs of fuels, gut hormones, and neurotransmitters, and the transduction of these inputs into signals that modulate secretion (Zawalich and Rasmussen, 1990, Bolaffi et al., 1991, Metz, 1991, Newgard and McGarry, 1995, Sharp, 1996). Phosphoinositide-specific phospholipase C (PLC) has been postulated to play an important role in the regulation of insulin secretion by these diverse secretagogues (Best and Malaisse, 1983, Best and Malaisse, 1984, Kelley et al., 1995, Zawalich et al., 1995b). In previous studies, we and others have provided insights into the pathway(s) that mediate glucose and hormonal stimulation of PLC (Best and Malaisse, 1983, Best and Malaisse, 1984, Montague et al., 1985, Biden et al., 1987, MacDonald et al., 1989, Yamaguchi et al., 1989, Gembal et al., 1993, Kelley et al., 1994, Kelley et al., 1995, Zawalich et al., 1995b); however, the exact signaling mechanism and the specific isozyme involved are not known.
In other tissues, 10 isoforms of PLC have been cloned which can be grouped into three main classes: PLC γ1–2, PLC β1–4 and PLC δ1–4 [for review, see Exton (1997) and Noh et al. (1995)]. PLC γ is activated by tyrosine phosphorylation by receptor and cytosolic tyrosine kinases (Noh et al., 1995); and PLC β is activated by heterotrimeric G-protein α subunits of the Gq family and by G-protein βγ subunits (Exton, 1997). The mechanism of activation of PLC δ is less well understood but has been shown to be regulated by changes in [Ca2+]i (Banno et al., 1994), G-proteins (Banno et al., 1994), GTP (Murthy et al., 1999), and the RhoGap p122 (Sekimata et al., 1999).
In the beta cell, each of the three main classes of PLC have been identified (Kelley et al., 1995, Gasa et al., 1999). As in other tissues, it is likely that heterotrimeric G-protein coupled receptor hormones that regulate PLC activity in the islet, such as vasopressin, cholecystokinin, and acetylcholine, activate the β class of PLC (Exton, 1997); although, the specific isoform, PLC β1–4, coupled to each agonist is not known.
Unlike hormone receptors, glucose and other fuels do not appear to bind to a receptor but generate signals during their metabolism to activate effectors (Newgard and McGarry, 1995). The pathway by which glucose stimulates PLC is distinct from hormone-receptor G-protein activation of PLC (Kelley et al., 1994, Kelley et al., 1995). Unlike hormonal activation of PLC, glucose stimulation is partially dependent on Ca2+ influx and additional metabolically generated signals (Yamaguchi et al., 1989, Kelley et al., 1994). In addition, maximal concentrations of glucose and the hormone carbachol or cholecystokinin interact synergistically to increase inositol phosphate (IP) accumulation in rat islets (Kelley et al., 1995). Furthermore, studies comparing glucose and hormone regulation of PLC in rat and mouse islets demonstrate unique activation mechanisms (Zawalich et al., 1995b): carbachol stimulates PLC equally well in rat and mouse islets; however, glucose stimulation in mouse islets is markedly deficient compared to rat islets. Studies comparing the isoforms of PLC present in these two species demonstrate a deficiency of PLC δ1 and PLC β1 in mouse islets compared to rat islets suggesting that glucose may regulate one or both of these two isoforms.
The purpose of these studies was to determine directly if fuels couple to PLC β1 and/or PLC δ1 in a beta cell line. Since fuels require intact cells to generate their signals, our approach was to overexpress these isoforms in the insulin secreting beta cell line RINm5F, and determine their regulation by fuels and hormones. These studies demonstrate a differential regulation of specific isoforms of PLC by diverse secretagogues in this beta cell line.
Section snippets
Tissue culture
Dr. Geoffrey Sharp (Cornell University, Ithaca, NY) provided RINm5F cells (passage 56). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 U/ml penicillin and 50 μg/ml streptomycin in a humidified 5% CO2 incubator at 37°C.
Stable transfections
Rat cDNAs for PLC β1 and PLC δ1 were obtained from Dr. Sue Goo Rhee (NIH, Bethesda, MD) and cloned into the expression vector pneoSRαII obtained from Dr. Alfred Bothwell (Yale University, New Haven, CT). RINm5F cells were seeded at a density of
Overexpressed PLC isozymes in RINm5F cells
After transfection with PLC β1 and PLC δ1 cDNA, G418 resistant clones were examined for overexpression of PLC isozymes. Of the clones isolated, only 1 out of 23 clones demonstrated overexpression of PLC β1, labeled β22, and 1 out of 16 clones demonstrated overexpression of PLC δ1, labeled δ5. One other PLC β1 and one PLC δ1 clone demonstrated overexpression of abnormal sized bands by Western blot analysis. A vector clone, V18, was randomly selected as a control. Additional studies demonstrated
Discussion
While hormone activation of PLC has been extensively characterized in many tissues (Noh et al., 1995, Exton, 1997), the mechanism by which glucose and other fuels stimulate this effector is not known. Evidence, however, demonstrates that hormones and fuels stimulate PLC by distinct pathways (Yamaguchi et al., 1989, Kelley et al., 1994, Kelley et al., 1995), and studies comparing activation of PLC in mouse and rat islets suggest that fuels couple to PLC β1 and PLC δ1 isoforms (Zawalich et al.,
Acknowledgements
Preliminary accounts of this work have appeared in abstract form.1 These studies were supported by grants from NIDDK 02089 and 45735 (Yale Diabetes Endocrinology Research Center), and the American Heart Association, New York Affiliate 9706336A.
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