Regulation of hepatic glucose production and AMPK by AICAR but not by metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1)

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Aim: Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate that besides activating protein kinase A, 8CPT-modified cAMP analogues suppress the Nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter ENT1. We aimed to exploit 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR, which is highly selective for a high-affinity binding-site on ENT1, to investigate the role of ENT1 in the liver specific glucose lowering properties of AICAR and metformin. Methods: Primary mouse hepatocytes were incubated with AICAR and metformin in combination with cAMP analogues, glucagon, forskolin and NBMPR. Hepatocyte glucose production (HGP), and AMPK signalling were measured and a uridine uptake assay with supporting LC-MS was used to investigate nucleoside depletion from medium by cells. Results: AICAR and metformin increased AMPK pathway phosphorylation and decreased HGP induced by dibutyryl cAMP and glucagon. HGP was also induced by 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR; however, in each case this was resistant to suppression by AICAR but not metformin. Cross-validation of tracer and mass spectrometry studies indicates that 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR inhibited the effects of AICAR at least in part by impeding its uptake into hepatocytes. Conclusions: We report for the first time that suppression of ENT1 induces HGP. ENT1 inhibition also impedes uptake and effects of AICAR but not metformin on HGP. Further investigation of nucleoside transport may illuminate a better understanding of how metformin and AICAR each regulate HGP.L.L. was supported by a Cunningham Trust PhD studentship awarded to G.R. and C.B. G.R. acknowledges additional support from the MRC (MR/K012924/1). This work was part-funded by a Tenovus Scotland grant to C.B, who is a recipient of a Diabetes UK RD Lawrence Fellowship (13/0004647)

Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase.

Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P2), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects

The emerging role of AMPK in the regulation of breathing and oxygen supply

Regulation of breathing is critical to our capacity to accommodate deficits in oxygen availability and demand during, for example, sleep and ascent to altitude. It is generally accepted that a fall in arterial oxygen increases afferent discharge from the carotid bodies to the brainstem and thus delivers increased ventilatory drive, which restores oxygen supply and protects against hypoventilation and apnoea. However, the precise molecular mechanisms involved remain unclear. We recently identified as critical to this process the AMP-activated protein kinase (AMPK), which is key to the cell-autonomous regulation of metabolic homoeostasis. This observation is significant for many reasons, not least because recent studies suggest that the gene for the AMPK-α1 catalytic subunit has been subjected to natural selection in high-altitude populations. It would appear, therefore, that evolutionary pressures have led to AMPK being utilized to regulate oxygen delivery and thus energy supply to the body in the short, medium and longer term. Contrary to current consensus, however, our findings suggest that AMPK regulates ventilation at the level of the caudal brainstem, even when afferent input responses from the carotid body are normal. We therefore hypothesize that AMPK integrates local hypoxic stress at defined loci within the brainstem respiratory network with an index of peripheral hypoxic status, namely afferent chemosensory inputs. Allied to this, AMPK is critical to the control of hypoxic pulmonary vasoconstriction and thus ventilation–perfusion matching at the lungs and may also determine oxygen supply to the foetus by, for example, modulating utero-placental blood flow

Juror gender and influence in a domestic violence trial

In two online mock jury deliberation studies (N1 = 326, N2 = 531), I investigate how a juror's gender and emotion expression impacts how influential they are perceived to be and how much influence they actually have in deliberation. Participants perceived a female juror to be less influential than a male juror, but were actually influenced by this juror equally regardless of gender. Neither form of influence was affected by the juror's emotion expression. I argue that because the trial depicted domestic violence, which is culturally associated with female victims, women have more influence in this situation than they typically would. These findings suggest that even when women are influential in interaction, they may not be acknowledged as such. Further, they highlight the importance of task content both theoretically in studies of status and gender and practically in contexts such as jury selection, particularly when that content includes gender-based violence