Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin-induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser79 and Raptor at Ser792, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05–0.1 mM) that were 10 to 100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α1 and α2 catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G q -coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3-10 nM) or with vasopressin, a different G q -coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser 744 and Ser 748 . Transphosphorylation targeted Ser 744 , whereas autophosphorylation was the predominant mechanism for Ser 748 in cells stimulated with G q -coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G q -coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G q -coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation requires the identification of the molecular pathways that govern the transition of quiescent cells into the S phase of the cell cycle. In this context the activation and phosphorylation of protein kinase D (PKD), 4 the founding member of a new protein kinase family within the Ca 2ϩ /calmodulin-dependent protein kinase (CAMK) group and separate from the previously identified PKCs (for review, see Ref. 1), are attracting intense attention. In unstimulated cells, PKD is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing a repeat of cysteinerich zinc finger-like motifs and a pleckstrin homology (PH) domain (1-4). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (5-7). In response to cellular stimuli (1), including phorbol esters, growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR) agonists (6, 8 -16) that signal through G q , G 12 , G i , and Rho...

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G q -coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G q -coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. The mammalian intestine is covered by a single layer of epithelial cells that is renewed every 4 -5 days along the cryptvillus axis (1). The high rate of cell turnover, driven by crypt cell proliferation, plays a fundamental role in the organization, maintenance, and restoration of tissue integrity. It is recognized that the sequential proliferation, lineage-specific differentiation, crypt-villus migration, and cell death of the epithelial cells of the intestinal mucosa is a tightly regulated process modulated by a broad range of regulatory peptides, differentiation signals, and luminal stimuli, including nutrients and pathogenic/commensal organisms (1-3). Despite its importance for understanding normal homeostasis, wound healing, and the pathogenesis of human disease states, including inflammatory bowel diseases and colon cancer, the intracellular signal transduction mechanisms involved remain incompletely understood.Protein kinase D1 (PKD1), 2 the founding member of a new protein kinase family within the calcium/calmodulin-dependent protein kinase group and separate from the previously identified PKCs (for review, see Ref. 4), is attracting intense attention. PKD1 has been extensively studied in vitro with regard to identifying the functions of its domains and the effect of cell signaling on its activity and subcellular localization (4). In unstimulated cells, PKD1 is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing a repeat of cysteinerich zinc finger-like motifs and a pleckstrin homology domain (4 -7). PKD1 can be activated within intact cells by multiple stimuli acting through receptor-mediated pathways (for review, see Ref. 4). Our own studies demonstrated rapid, PKCdependent, PKD1 activation in response to G protein-coupled receptor (GPCR) agonists, including regulatory peptides (8 -17) and bioactive lipids (12, 18 -20) that act through G q , G 12 , G i , and Rho (12,[17][18][19]21,22), growth factors that act though tyrosine-kinase receptors (8, 23), cross-linking of Bcell receptor, and T-cell receptor in B and T lymphocytes (24 -26) and oxidative stress (27,28). The phosphorylation of Ser 744 and Ser 748 in the PKD1 activation loop (also referred as activation segment or T-loop) is critical for PKD1 activation (4,...