Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of ␤-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of ␤-amyloid (A␤) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage R ), affects APP metabolism and A␤ generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular A␤ species. Furthermore, the effect of metformin on A␤ generation is mediated by transcriptional up-regulation of ␤-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on A␤ degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on A␤. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on A␤ generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on A␤ generation, in combined use, metformin enhances insulin's effect in reducing A␤ levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients. A lzheimer's disease (AD) is a devastating neurodegenerative disorder, with aging, genetic, and environmental factors contributing to its development and progression. AD is not only characterized by pathological deposition of A␤ peptides and neurofibrillary tangles but is also associated with microgliamediated inflammation and dysregulated lipid homeostasis and glucose metabolism. Amyloid peptides are derived from sequential proteolytic cleavages of full-length amyloid precursor protein (APP) by ␤-secretase (BACE1) and ␥-secretase. Full-length APP can undergo alternative processing by ␣-secretase, releasing a soluble fragment (sAPP␣) extracellularly, which precludes A␤ formation. Compelling evidence indicates that A␤, especially the oligomers, are toxic to neurons; excessive generation and accumulation of A␤ peptides in neurons is believed to initiate the pathological cascade in AD (1-3).Epidemiological studies strongly suggest that metabolic defects correlate with the functional alterations associated with aging of the brain and with AD pathogenesis (4-11). The vast majority of AD cases are late onset and sporadic in origin with aging being the most profound risk factor. Insulin signaling is known to be involved in the process of brain aging (12)(13)(14)(15)(16)(17)(18)(19)(20). Insulin dysfunction/resistance in diabetes mellitus (DM) is not only a common syndrome ...

cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37) and the 14-3-3 proteins. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin. These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues, and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially. The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain and is investigated here. Active Raf can be almost completely deactivated in vitro by displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. We propose that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.

The c-Raf-1 proto-oncoprotein is a Ras-GTP-regulated protein kinase that associates in situ with 14-3-3 proteins, which are naturally dimeric. In COS cells, recombinant Raf is found in oligomeric assemblies. To examine whether induced oligomerization can alter Raf kinase activity, sequences encoding the FK506-binding protein FKBP12 were fused to the amino terminus of c-Raf-1, introducing a binding site for FK506. Oligomerization of recombinant FKBP-Raf in situ, induced by the addition of the dimeric FK506 derivative FK1012A, activated Raf kinase activity at least half as well as epidermal growth factor (EGF). As with EGF, activation of FKBP-Raf by FK1012A is entirely Ras-GTP dependent. Thus oligomerization of Raf per se promotes Raf activation through a Ras-dependent mechanism.