Metformin is a first-line therapeutic option for the treatment of type 2 diabetes, even though its underlying mechanisms of action are relatively unclear. Metformin lowers blood glucose levels by inhibiting hepatic glucose production (HGP), an effect originally postulated to be due to a hepatic AMP-activated protein kinase (AMPK)-dependent mechanism. However, studies have questioned the contribution of hepatic AMPK to the effects of metformin on lowering hyperglycemia, and a gut-brain-liver axis that mediates intestinal nutrient- and hormone-induced lowering of HGP has been identified. Thus, it is possible that metformin affects HGP through this inter-organ crosstalk. Here we show that intraduodenal infusion of metformin for 50 min activated duodenal mucosal Ampk and lowered HGP in a rat 3 d high fat diet (HFD)-induced model of insulin resistance. Inhibition of duodenal Ampk negated the HGP-lowering effect of intraduodenal metformin, and both duodenal glucagon-like peptide-1 receptor (Glp-1r)-protein kinase A (Pka) signaling and a neuronal-mediated gut-brain-liver pathway were required for metformin to lower HGP. Preabsorptive metformin also lowered HGP in rat models of 28 d HFD-induced obesity and insulin resistance and nicotinamide (NA)-streptozotocin (STZ)-HFD-induced type 2 diabetes. In an unclamped setting, inhibition of duodenal Ampk reduced the glucose-lowering effects of a bolus metformin treatment in rat models of diabetes. These findings show that, in rat models of both obesity and diabetes, metformin activates a previously unappreciated duodenal Ampk-dependent pathway to lower HGP and plasma glucose levels.

The LKB1 tumor suppressor is a protein kinase that controls activity of adenine monophosphateactivated protein kinase (AMPK). LKB1 activity is regulated by the pseudokinase STRADα and the scaffolding protein MO25α, through an unknown, phosphorylation-independent, mechanism. We describe the structure of the core heterotrimeric LKB1-STRADα-MO25α complex, revealing an unusual allosteric mechanism of LKB1 activation. STRADα adopts a closed conformation typical of active protein kinases and binds LKB1 as a pseudosubstrate. STRADα and MO25α promote the active conformation of LKB1, which is stabilised by MO25α interacting with the LKB1 activation loop. This previously undescribed mechanism of kinase activation may be relevant to understanding the evolution of other pseudokinases. The structure also reveals how mutations found in Peutz-Jeghers syndrome and other cancers impair LKB1 function.Loss-of-function mutations in the LKB1 tumor suppressor gene cause the rare inherited disease Peutz-Jeghers cancer Syndrome (PJS) in humans [1] and are associated with various sporadic cancers, in particular non-small cell lung cancer (NSCLC) [2]. One prominent function of LKB1 is to ensure that growth and division are coupled to the availability of cellular energy. LKB1 phosphorylates and activates the adenosine monophosphate (AMP)-activated protein kinase (AMPK) when energy levels are low, thereby leading to inhibition of signalling pathways that promote proliferation [3]. The therapeutic effects of AMPKactivating drugs (e.g. metformin) on reducing tumor growth [4], or blood glucose levels [5] are dependent on activation of AMPK by LKB1. Another key role of LKB1 is to control cell polarity, which may be mediated by AMPK [6] as well as a group of AMPK-related protein kinases, including microtubule affinity regulating kinases (MARKs, homologous to the C. elegans kinase Par-1) [7] that are also phosphorylated and activated by LKB1 [8].In cells, LKB1 is found in a 1:1:1 heterotrimeric complex with the pseudokinase STRAD (STe20-Related ADaptor) [9] and the scaffolding MO25 (MOuse protein 25) [10]. There are two closely related human isoforms of both STRAD (STRADα and STRADβ) and MO25 (MO25α and MO25β) that similarly interact with LKB1 [11]. Unlike the majority of protein kinases, which are regulated by phosphorylation, LKB1 is activated by binding to STRAD and MO25 [12,11] through an unknown, phosphorylation-independent, molecular mechanism. Structural analysis of MO25α reveals a helical repeat, horseshoe-shaped protein, that interacts with the C-terminal WEF (Trp-Glu-Phe) motif of STRADα through a hydrophobic pocket, located on its convex C-terminal surface [13]. The structure of

Mouse protein-25 (MO25) isoforms bind to the STRAD pseudokinase and stabilise it in a conformation that can activate the LKB1 tumour suppressor kinase. We demonstrate that by binding to several STE20 family kinases, MO25 has roles beyond controlling LKB1. These new MO25 targets are SPAK/OSR1 kinases, regulators of ion homeostasis and blood pressure, and MST3/MST4/YSK1, involved in controlling development and morphogenesis. Our analyses suggest that MO25a and MO25b associate with these STE20 kinases in a similar manner to STRAD. MO25 isoforms induce approximately 100-fold activation of SPAK/OSR1 dramatically enhancing their ability to phosphorylate the ion cotransporters NKCC1, NKCC2 and NCC, leading to the identification of several new phosphorylation sites. siRNA-mediated reduction of expression of MO25 isoforms in mammalian cells inhibited phosphorylation of endogenous NKCC1 at residues phosphorylated by SPAK/OSR1, which is rescued by re-expression of MO25a. MO25a/b binding to MST3/MST4/YSK1 also stimulated kinase activity three-to four-fold. MO25 has evolved as a key regulator of a group of STE20 kinases and may represent an ancestral mechanism of regulating conformation of pseudokinases and activating catalytically competent protein kinases.

The conformation of the pseudokinase STRADα, which is regulated by binding to ATP and to the scaffolding protein MO25α, is key to the activiation of the LKB1 tumor suppressor complex.

Insulin activates PI3-kinase (PI3K)/AKT to regulate glucose homeostasis in the peripheral tissues and the mediobasal hypothalamus (MBH) of rodents. We report that insulin infusion into the MBH or dorsal vagal complex (DVC) activated insulin receptors. The same dose of insulin that activated MBH PI3K/AKT did not in the DVC. DVC insulin instead activated Erk1/2 and lowered glucose production in rats and mice. Molecular and chemical inhibition of DVC Erk1/2 negated, while activation of DVC Erk1/2 recapitulated, the effects of DVC insulin. Circulating insulin failed to inhibit glucose production when DVC Erk1/2 was inhibited in normal rodents, while DVC insulin action was disrupted in high-fat-fed rodents. Activation of DVC ATP-sensitive potassium channels was necessary for insulin-Erk1/2 and sufficient to inhibit glucose production in normal and high-fat-fed rodents. DVC is a site of insulin action where insulin triggers Erk1/2 signaling to inhibit glucose production and of insulin resistance in high-fat feeding.

Glucagon activates hepatic protein kinase A (PKA) to increase glucose production, but the gluco-stimulatory effect is transient even in the presence of continuous intravenous glucagon infusion. Continuous intravenous infusion of insulin, however, inhibits glucose production through its sustained actions in both the liver and the mediobasal hypothalamus (MBH). In a pancreatic clamp setting, MBH infusion with glucagon activated MBH PKA and inhibited hepatic glucose production (HGP) in rats, as did central glucagon infusion in mice. Inhibition of glucagon receptor-PKA signaling in the MBH and hepatic vagotomy each negated the effect of MBH glucagon in rats, whereas the central effect of glucagon was diminished in glucagon receptor knockout mice. A sustained rise in plasma glucagon concentrations transiently increased HGP, and this transiency was abolished in rats with negated MBH glucagon action. In a nonclamp setting, MBH glucagon infusion improved glucose tolerance, and inhibition of glucagon receptor-PKA signaling in the MBH enhanced the ability of intravenous glucagon injection to increase plasma glucose concentrations. We also detected a similar enhancement of glucose concentrations that was associated with a disruption in MBH glucagon signaling in rats fed a high-fat diet. We show that hypothalamic glucagon signaling inhibits HGP and suggest that hypothalamic glucagon resistance contributes to hyperglycemia in diabetes and obesity.

Resveratrol improves insulin sensitivity and lowers hepatic glucose production (HGP) in rat models of obesity and diabetes, but the underlying mechanisms for these antidiabetic effects remain elusive. One process that is considered a key feature of resveratrol action is the activation of the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase sirtuin 1 (SIRT1) in various tissues. However, the low bioavailability of resveratrol raises questions about whether the antidiabetic effects of oral resveratrol can act directly on these tissues. We show here that acute intraduodenal infusion of resveratrol reversed a 3 d high fat diet (HFD)-induced reduction in duodenal-mucosal Sirt1 protein levels while also enhancing insulin sensitivity and lowering HGP. Further, we found that duodenum-specific knockdown of Sirt1 expression for 14 d was sufficient to induce hepatic insulin resistance in rats fed normal chow. We also found that the glucoregulatory role of duodenally acting resveratrol required activation of Sirt1 and AMP-activated protein kinase (Ampk) in this tissue to initiate a gut-brain-liver neuronal axis that improved hypothalamic insulin sensitivity and in turn, reduced HGP. In addition to the effects of duodenally acting resveratrol in an acute 3 d HFD-fed model of insulin resistance, we also found that short-term infusion of resveratrol into the duodenum lowered HGP in two other rat models of insulin resistance--a 28 d HFD-induced model of obesity and a nicotinamide (NA)-streptozotocin (STZ)-HFD-induced model of mild type 2 diabetes. Together, these studies highlight the therapeutic relevance of targeting duodenal SIRT1 to reverse insulin resistance and improve glucose homeostasis in obesity and diabetes.

The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.