A Role for Gz in Pancreatic Islet β-Cell Biology

Abstract

Glucose-stimulated insulin secretion and ␤-cell growth are important facets of pancreatic islet ␤-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G z and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic ␤-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G␣ z impacts are intact in ␤-cells, and G␣ z activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) ␤-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G␣ i family members are not involved in this process in this ␤-cell line. Indeed, overexpression of a selective deactivator of G␣ z , the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucosestimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G␣ z expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) ␤-cell line couples to G z predominantly and perhaps even exclusively. These data provide the first evidence for G z signaling in pancreatic ␤-cells, and identify an endogenous receptormediated signaling process in ␤-cells that is dependent on G␣ z function.Pancreatic islet ␤-cells are the sole producers of insulin in the body and thus play an important homeostatic role in the secretion of insulin in response to glucose and other secretagogues. Normally, the release of insulin from ␤-cells induces the peripheral tissues to take up available glucose from the plasma. Disregulation of this process can lead to the development of Type 2 diabetes, wherein the ␤-cells are unable to compensate for peripheral tissue insulin resistance, either through inadequate ␤-cell function and/or a decrease in ␤-cell mass (1).Certain hormones such as norepinephrine, somatostatin, galanin, and E prostaglandins decrease the secretion of insulin from ␤-cells in response to glucose (2). Pretreatment of pancreatic islet preparations with pertussis toxin (PTX) 1 alleviates the inhibitory effects of some of these hormones, leading to the original description of PTX as islet-activating protein (3). PTX catalyzes the ADP-ribosylation of a cysteine residue near the C terminus of nearly all G␣ subunits of the G␣ i subfamily (4), suggesting that the inhibition of glucose-stimulated insulin secretion (GSIS) is mediated by G␣ i proteins. However, several reports indicate a lack of effect, or only a partial effect, of PTX pretreatment on inhibition of insulin secretion in islets (5-8). This phenomenon of PTX insensitivity has been suggested to be because of insufficient PTX or too short a period of exposure (2), although an alternativ...

Evolving insights regarding mechanisms for the inhibition of insulin release by norepinephrine and heterotrimeric G proteins

“…Also completely novel was the finding that the PTX-insensitive heterotrimeric G protein Gz mediates the effect. While Gz has not previously been linked to norepinephrine, it is present in the ␤-cell and is activated by PGE 1 (57,58). The probable reason why this effect of norepinephrine has been hidden for so long is that as norepinephrine inhibits exocytosis, there was little need to look for an effect on endocytosis.…”

Gαz Negatively Regulates Insulin Secretion and Glucose Clearance

“…Immunohistochemical Assays-Tissues were dissected, fixed, and sectioned as previously described (13). For insulin immunostaining, the HistoMouse-MAX kit (Invitrogen) was used with prediluted guinea pig anti-insulin primary antibody (Invitrogen) incubated for 1 h at room temperature.…”

“…As expected for a member of the G␣ i subfamily, constitutively activated G␣ z inhibited GSIS from 832/13 cells (13). More importantly, inhibition of insulin secretion by prostaglandin E1 was PTX-insensitive and could be blocked either by overexpression of the "regulator of G protein signaling" domain of RGSZ1, a G␣ z -specific deactivator (15), or by small interfering RNA-mediated knockdown of G␣ z expression (13). Together, these data indicated that endogenous G␣ z in the 832/13 cells coupled to endogenous G proteincoupled receptors to inhibit insulin secretion.…”

“…Other unique G␣ z characteristics are a very low GDP-to-GTP exchange rate that is reduced 20-fold further in the presence of physiologic concentrations of Mg 2ϩ and an extremely low intrinsic GTP hydrolysis rate that is 200-fold lower than that described for other G␣ subunits (11). G␣ z also displays a limited expression profile, and protein expression has been demonstrated conclusively only in the brain, adrenal medulla, retina, platelets, pituitary, and pancreatic islets (10,12,13).…”

Prostaglandin E1 inhibits endocytosis in the β-cell endocytosis

“…The mechanism involves G βγ blockade of syntaxin binding to SNAP25 , Gerachshenko et al 2005, Photowala et al 2006. Although it was once believed that all physiological inhibitory effects on the β-cells were mediated by the Pertussis toxinsensitive (PTX)-sensitive heterotrimeric G i and G o proteins (Komatsu et al 1995), it has been reported that inhibition of adenylyl cyclase by PGE 1 is mediated via G z (Kimple et al 2005(Kimple et al , 2008. Exocytosis has to be followed by endocytosis to recapture the granule membrane that has been added to the plasma membrane of the cell in order to maintain cellular homeostasis and integrity (Rizzoli & Jahn 2007).…”