Inhibition of glucose-stimulated activation of extracellular signal-regulated protein kinases 1 and 2 by epinephrine in pancreatic beta-cells.

Glucose sensing is essential for the ability of pancreatic beta-cells to produce insulin in sufficient quantities to maintain blood glucose within the normal range. Stress causes the release of adrenergic hormones that increase circulating glucose by promoting glucose production and inhibiting insulin release. We have shown that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are responsive to glucose in pancreatic beta-cells and that glucose activates ERK1/2 by mechanisms independent of insulin. Here we show that glucose-induced activation of ERK1/2 is inhibited by epinephrine through the alpha2-adrenergic receptor. Epinephrine and the selective alpha2-adrenergic agonist UK14304 reduced insulin secretion and glucose-stimulated ERK1/2 activation in a pertussis toxin-sensitive manner, implicating the alpha subunit of a Gi family member. Alpha2-adrenergic agonists also reduced stimulation of ERK1/2 by glucagon-like peptide 1 and KCl, but not by phorbol ester or nerve growth factor. Our findings suggest that alpha2-adrenergic agonists act via a Gi family member on early steps in ERK1/2 activation, supporting the idea that ERK1/2 are regulated in a manner that reflects insulin demand.

MAP kinases and their roles in pancreatic beta-cells.

We discuss our work examining regulation and functions of mitogen-activated protein kinases, particularly ERK1 and ERK2, in pancreatic beta-cells. These enzymes are activated by glucose, other nutrients, and insulinogenic hormones. Their activation by these agents is calcium-dependent. A number of other stimuli also activate ERK1/2, but by mechanisms distinct from those involved in nutrient sensing. Inhibition of ERK1/2 has no apparent effect on insulin secretion measured after 2 h. On the other hand, ERK1/2 activity is required for maximal glucose-dependent activation of the insulin gene promoter. The primary effort has focused on INS-1 cell lines, with supporting and confirmatory studies in intact islets and other beta-cell lines, indicating the generality of our findings in beta-cell function. Thus ERK1/2 participate in transmitting glucose-sensing information to beta-cell functions. These kinases most likely act directly and indirectly on multiple pathways that regulate beta-cell function and, in particular, to transduce an elevated glucose signal into insulin gene transcription.

Regulation of ERK1 and ERK2 by glucose and peptide hormones in pancreatic beta cells.

We showed previously that ERK1/2 were activated by glucose and amino acids in pancreatic beta cells. Here we examine and compare signaling events that are necessary for ERK1/2 activation by glucose and other stimuli in beta cells. We find that agents that interrupt Ca2+ signaling by a variety of mechanisms interfere with glucose- and glucagon-like peptide (GLP-1)-stimulated ERK1/2 activity. In particular, calmodulin antagonists, FK506, and cyclosporin, immunosuppressants that inhibit the calcium-dependent phosphatase calcineurin, suppress ERK1/2 activation by both glucose and GLP-1. Ca2+ signaling from intracellular stores is also essential for ERK1/2 activation, because thapsigargin blocks ERK1/2 activation by glucose or GLP-1. The glucose-sensitive mechanism is distinct from that used by phorbol ester or insulin to stimulate ERK1/2 but shares common features with that used by GLP-1.

p115 Rho GTPase activating protein interacts with MEKK1.

Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway. Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization. MEKK1 is activated by molecules that impact cytoskeletal function. MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility. MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins. Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP). The p115 Rho GAP binds to MEKK1 in vitro and in intact cells. The p115 Rho GAP has selectivity for RhoA over other Rho family members. Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1. The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity. Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization.