Charlotte Green

Molecular connexions between dementia and diabetes.

Recent evidence suggests that the molecular defects associated with the development of diabetes also contribute to an increased risk of all types of dementia, including Alzheimers disease, vascular dementia and Picks disease. Indeed, the presence of type II diabetes mellitus results in a two to three fold higher risk of developing dementia [Fontbonne et al., 2001. Changes in cognitive abilities over a 4-year period are unfavourably affected in elderly diabetic subjects: results of the Epidemiology of Vascular Aging Study. Diabetes Care 24, 366-370; Gregg et al., 2000. Is diabetes associated with cognitive impairment and cognitive decline among older women? Study of Osteoporotic Fractures Research Group. Archives of Internal Medicine 160, 174-180; Peila et al., 2002. Type 2 diabetes, APOE gene, and the risk for dementia and related pathologies: The Honolulu-Asia Aging Study. Diabetes 51, 1256-1262]. There are currently 250 million people worldwide (>2 million in the UK) diagnosed with diabetes, and this number is predicted to double within the next 20 years, therefore the associated risk translates into a potential explosion in the appearance of dementia in the population. This review primarily focuses on the proposed molecular links between insulin action, Diabetes and Alzheimers disease, while discussing the potential for therapeutic intervention to alleviate these disorders. In particular, we will review the regulation of glycogen synthase kinase-3 (GSK-3) and its neuronal substrates.

Use of Akt Inhibitor and a Drug-resistant Mutant Validates a Critical Role for Protein Kinase B/Akt in the Insulin-dependent Regulation of Glucose and System A Amino Acid Uptake

Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKBα and -β was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti did not inhibit IRS1 tyrosine phosphorylation, phosphatidylinositol 3-kinase signaling, or activation of Erks, ribosomal S6 kinase, or atypical protein kinases C but significantly impaired regulation of downstream PKB targets glycogen synthase kinase-3 and AS160. Akti-mediated inhibition of PKB requires an intact kinase pleckstrin homology domain but does not involve suppression of 3-phosphoinositide binding to this domain. Importantly, we have discovered that Akti inhibition is critically dependent upon a solvent-exposed tryptophan residue (Trp-80) that is present within the pleckstrin homology domain of all three PKB isoforms and whose mutation to an alanine (PKBW80A) yields an Akti-resistant kinase. Cellular expression of PKBW80A antagonized the Akti-mediated inhibition of glucose and amino acid uptake. Our findings support a critical role for PKB in the hormonal regulation of glucose and system A amino acid uptake and indicate that use of Akti and expression of the drug-resistant kinase will be valuable tools in delineating cellular PKB functions.

Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells.

Background: The role of AMPK and PKCs as effectors of metformin action on glucose uptake (GU) in skeletal muscle cells was investigated. Results: Genetic loss/silencing of AMPK led to only a small repression in metformin-stimulated GU. Novel/conventional, but not atypical, PKCs support metformin-induced stimulation of GU. Conclusion: Metformin enhances GU by a mechanism largely independent of AMPK. Significance: Metformin can act via non-AMPK pathways to promote GU. The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ∼20%. Consistent with this, analysis of GU in muscle cells from α1−/−/α2−/− AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ∼35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.

The PPARδ agonist, GW501516, promotes fatty acid oxidation but has no direct effect on glucose utilisation or insulin sensitivity in rat L6 skeletal muscle cells

Peroxisome proliferator‐activated receptor‐delta (PPARδ) activation enhances skeletal muscle fatty acid oxidation and improves whole body glucose homeostasis and insulin sensitivity. Recently, GW501516, a selective PPARδ agonist, was reported to increase glucose uptake in human skeletal myotubes by an AMPK‐dependent mechanism that may contribute to the improved glucose tolerance. Here, we demonstrate that whilst GW501516 increases expression of PGC‐1α and CPT‐1 and stimulates fatty‐acid oxidation in L6 myotubes, it fails to enhance insulin sensitivity, AMPK activity or glucose uptake and storage. Our findings exclude sarcolemmal glucose transport as a potential target for the therapeutic action of PPARδ agonists in skeletal muscle.

Pyruvate suppresses PGC1α expression and substrate utilization despite increased respiratory chain content in C2C12 myotubes

Sodium pyruvate can increase mitochondrial biogenesis in C2C12 myoblasts in a peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α)-independent manner. The present study examined the effect of 72-h treatment with sodium pyruvate (5–50 mM) or sodium chloride (50 mM) as an osmotic control on the regulation of mitochondrial substrate metabolism and biogenesis in C2C12 myotubes. Pyruvate (50 mM) increased the levels of fatty acid oxidation enzymes (CD36, 61%, and β-oxidative enzyme 3-hydroxyacyl-CoA dehydrogenase, 54%) and the expression of cytochrome-c oxidase subunit I (220%) and cytochrome c (228%), consistent with its previous described role as a promoter of mitochondrial biogenesis. However, in contrast, pyruvate treatment reduced glucose transporter 4 (42%), phosphofructokinase (57%), and PGC1α (72%) protein content as well as PGC1α (48%) and PGC1β (122%) mRNA. The decrease in PGC1α was compensated for by an increase in the PGC1α-related coactivator (PRC; 187%). Pyruvate treatment reduced basal and insulin-stimulated glucose uptake (41% and 31%, respectively) and palmitate uptake and oxidation (24% and 31%, respectively). The addition of the pyruvate dehydrogenase activator dichloroacetate (DCA) and the TCA precursor glutamine increased PGC1α expression (368%) and returned PRC expression to basal. Glucose uptake increased by 4.2-fold with DCA and glutamine and palmitate uptake increased by 18%. Coupled to this adaptation was an 80% increase in oxygen consumption. The data suggest that supraphysiological doses of pyruvate decrease mitochondrial function despite limited biogenesis and that anaplerotic agents can reverse this effect.