Associations of neighborhood environment with brain imaging outcomes in the Australian Imaging, Biomarkers and Lifestyle cohort.

INTRODUCTION: "Walkable" neighborhoods offer older adults opportunities for activities that may benefit cognition-related biological mechanisms. These have not previously been examined in this context. METHODS: We objectively assessed neighborhood walkability for participants (n = 146) from the Australian Imaging, Biomarkers and Lifestyle study with apolipoprotein E (APOE) genotype and two 18-month-apart brain volumetric and/or amyloid ß burden assessments. Linear mixed models estimated associations of neighborhood walkability with levels and changes in brain imaging outcomes, the moderating effect of APOE ε4 status, and the extent to which associations were explained by physical activity. RESULTS: Cross-sectionally, neighborhood walkability was predictive of better neuroimaging outcomes except for left hippocampal volume. These associations were to a small extent explained by physical activity. APOE ε4 carriers showed slower worsening of outcomes if living in walkable neighborhoods. DISCUSSION: These findings indicate associations between neighborhood walkability and brain imaging measures (especially in APOE ε4 carriers) minimally attributable to physical activity.

Innate phagocytosis by peripheral blood monocytes is altered in Alzheimer's disease.

Sporadic Alzheimer's disease (AD) is characterised by the deposition and accumulation of specific protein aggregates. Failure of clearance could underlie this process, and recent genetic association studies point towards involvement of the phagocytosis and autophagy pathways. We developed a real-time tri-color flow cytometry method to quantitate the phagocytic function of human peripheral blood monocyte subsets including non-classic CD14(dim)CD16(+), intermediate CD14(+)CD16(+) and classic CD14(+)CD16(-) monocytes. Using this method, we have measured the phagocytic ability of fresh monocytes in a study of preclinical, prodromal and clinical AD, matched with cognitively normal healthy control subjects. Basal levels of phagocytosis in all three subsets of monocytes were similar between healthy controls and AD patients, while a significant increase of basal phagocytosis was found in subjects with high Aß-amyloid burden as assessed by PET scans. Pre-treating cells with Copaxone (CPX, to stimulate phagocytosis) or ATP (an inhibitor of P2X7-mediated phagocytosis) showed a differential response depending on clinical or Aß-burden status, indicating a relative functional deficit. Overall the results are consistent with a perturbation of basal and stimulated innate phagocytosis in sporadic AD.

Comparative analysis of the Cancer Council of Victoria and the online Commonwealth Scientific and Industrial Research Organisation FFQ.

FFQ are commonly used to examine the association between diet and disease. They are the most practical method for usual dietary data collection as they are relatively inexpensive and easy to administer. In Australia, the Cancer Council of Victoria FFQ (CCVFFQ) version 2 and the online Commonwealth Scientific and Industrial Research Organisation FFQ (CSIROFFQ) are used. The aim of our study was to establish the level of agreement between nutrient intakes captured using the online CSIROFFQ and the paper-based CCVFFQ. The CCVFFQ and the online CSIROFFQ were completed by 136 healthy participants. FFQ responses were analysed to give g per d intake of a range of nutrients. Agreement between twenty-six nutrient intakes common to both FFQ was measured by a variety of methods. Nutrient intake levels that were significantly correlated between the two FFQ were carbohydrates, total fat, Na and MUFA. When assessing ranking of nutrients into quintiles, on average, 56 % of the participants (for all nutrients) were classified into the same or adjacent quintiles in both FFQ, with the highest percentage agreement for sugar. On average, 21 % of participants were grossly misclassified by three or four quintiles, with the highest percentage misclassification for fibre and Fe. Quintile agreement was similar to that reported by other studies, and we concluded that both FFQ are suitable tools for dividing participants' nutrient intake levels into high- and low-consumption groups. Use of either FFQ was not appropriate for obtaining accurate estimates of absolute nutrient intakes.

Predicting Alzheimer disease with ß-amyloid imaging: results from the Australian imaging, biomarkers, and lifestyle study of ageing.

OBJECTIVE: Biomarkers for Alzheimer disease (AD) can detect the disease pathology in asymptomatic subjects and individuals with mild cognitive impairment (MCI), but their cognitive prognosis remains uncertain. We aimed to determine the prognostic value of ß-amyloid imaging, alone and in combination with memory performance, hippocampal atrophy, and apolipoprotein E ε4 status in nondemented, older individuals. METHODS: A total of 183 healthy individuals (age = 72.0 ± 7.26 years) and 87 participants with MCI (age = 73.7 ± 8.27) in the Australian Imaging, Biomarkers, and Lifestyle study of ageing were studied. Clinical reclassification was performed after 3 years, blind to biomarker findings. ß-Amyloid imaging was considered positive if the (11) C-Pittsburgh compound B cortical to reference ratio was ≥1.5. RESULTS: Thirteen percent of healthy persons progressed (15 to MCI, 8 to dementia), and 59% of the MCI cohort progressed to probable AD. Multivariate analysis showed ß-amyloid imaging as the single variable most strongly associated with progression. Of combinations, subtle memory impairment (Z score = -0.5 to -1.5) with a positive amyloid scan was most strongly associated with progression in healthy individuals (odds ratio [OR] = 16, 95% confidence interval [CI] = 3.7-68; positive predictive value [PPV] = 50%, 95% CI = 19-81; negative predictive value [NPV] = 94%, 95% CI = 88-98). Almost all amnestic MCI subjects (Z score ≤ -1.5) with a positive amyloid scan developed AD (OR = ∞; PPV = 86%, 95% CI = 72-95; NPV = 100%, 95% CI = 80-100). Hippocampal atrophy and ε4 status did not add further predictive value. INTERPRETATION: Subtle memory impairment with a positive ß-amyloid scan identifies healthy individuals at high risk for MCI or AD. Clearly amnestic patients with a positive amyloid scan have prodromal AD and a poor prognosis for dementia within 3 years.

Autobiographical narratives relate to Alzheimer's disease biomarkers in older adults.

BACKGROUND: Autobiographical memory (ABM), personal semantic memory (PSM), and autonoetic consciousness are affected in individuals with mild cognitive impairment (MCI) but their relationship with Alzheimer's disease (AD) biomarkers are unclear. METHODS: Forty-five participants (healthy controls (HC) = 31, MCI = 14) completed the Episodic ABM Interview and a battery of memory tests. Thirty-one (HC = 22, MCI = 9) underwent ß-amyloid positron emission tomography (PET) and magnetic resonance (MR) imaging. Fourteen participants (HC = 9, MCI = 5) underwent one imaging modality. RESULTS: Unlike PSM, ABM differentiated between diagnostic categories but did not relate to AD biomarkers. Personal semantic memory was related to neocortical ß-amyloid burden after adjusting for age and apolipoprotein E (APOE) ɛ4. Autonoetic consciousness was not associated with AD biomarkers, and was not impaired in MCI. CONCLUSIONS: Autobiographical memory was impaired in MCI participants but was not related to neocortical amyloid burden, suggesting that personal memory systems are impacted by differing disease mechanisms, rather than being uniformly underpinned by ß-amyloid. Episodic and semantic ABM impairment represent an important AD prodrome.

Rates of diagnostic transition and cognitive change at 18-month follow-up among 1,112 participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL).

BACKGROUND: The Australian Imaging, Biomarkers and Lifestyle (AIBL) Flagship Study of Ageing is a prospective study of 1,112 individuals (211 with Alzheimer's disease (AD), 133 with mild cognitive impairment (MCI), and 768 healthy controls (HCs)). Here we report diagnostic and cognitive findings at the first (18-month) follow-up of the cohort. The first aim was to compute rates of transition from HC to MCI, and MCI to AD. The second aim was to characterize the cognitive profiles of individuals who transitioned to a more severe disease stage compared with those who did not. METHODS: Eighteen months after baseline, participants underwent comprehensive cognitive testing and diagnostic review, provided an 80 ml blood sample, and completed health and lifestyle questionnaires. A subgroup also underwent amyloid PET and MRI neuroimaging. RESULTS: The diagnostic status of 89.9% of the cohorts was determined (972 were reassessed, 28 had died, and 112 did not return for reassessment). The 18-month cohort comprised 692 HCs, 82 MCI cases, 197 AD patients, and one Parkinson's disease dementia case. The transition rate from HC to MCI was 2.5%, and cognitive decline in HCs who transitioned to MCI was greatest in memory and naming domains compared to HCs who remained stable. The transition rate from MCI to AD was 30.5%. CONCLUSION: There was a high retention rate after 18 months. Rates of transition from healthy aging to MCI, and MCI to AD, were consistent with established estimates. Follow-up of this cohort over longer periods will elucidate robust predictors of future cognitive decline.

Enabling a multidisciplinary approach to the study of ageing and Alzheimer's disease: an update from the Australian Imaging Biomarkers and Lifestyle (AIBL) study.

The Australian Imaging Biomarkers and Lifestyle (AIBL) study is a longitudinal study of 1,112 volunteers from healthy, mild cognitive impairment (MCI) and Alzheimer's disease (AD) populations who are assessed at 18-month intervals in order to enable prospective research into ageing and AD. Using a multidisciplinary battery, AIBL assessments comprise the extensive study of clinical factors and cognitive function, collection of blood and cerebrospinal fluid (CSF) samples for biomarker discovery, structural and ß-amyloid (Aß) neuroimaging, and obtaining information on diet and physical activity patterns of the cohort. Now in its seventh year, AIBL is part of a substantial international effort to prospectively study the relationships between clinical characteristics and putative AD biomarkers in groups who carry different risk factors for AD. The identification of biomarkers would provide a window of opportunity to assess AD risk in individuals prior to the onset of advanced clinical symptoms, in addition to facilitating testing of therapeutic and lifestyle interventions likely to emerge within the next decade that prevent or delay symptom emergence in those at high risk for developing AD. In this paper, we present key findings from the AIBL study and discuss how they contribute to our understanding of AD pathogenesis and diagnosis.

ß-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK).

The AMP-activated protein kinase (AMPK) is an αßγ heterotrimer that acts as a master metabolic regulator to maintain cellular energy balance following increased energy demand and increases in the AMP/ATP ratio. This regulation provides dynamic control of energy metabolism, matching energy supply with demand that is essential for the function and survival of organisms. AMPK is inactive unless phosphorylated on Thr172 in the α-catalytic subunit activation loop by upstream kinases (LKB1 or calcium-calmodulin-dependent protein kinase kinase ß). How a rise in AMP levels triggers AMPK α-Thr172 phosphorylation and activation is incompletely understood. Here we demonstrate unequivocally that AMP directly stimulates α-Thr172 phosphorylation provided the AMPK ß-subunit is myristoylated. Loss of the myristoyl group abolishes AMP activation and reduces the extent of α-Thr172 phosphorylation. Once AMPK is phosphorylated, AMP further activates allosterically but this activation does not require ß-subunit myristoylation. AMP and glucose deprivation also promote membrane association of myristoylated AMPK, indicative of a myristoyl-switch mechanism. Our results show that AMP regulates AMPK activation at the initial phosphorylation step, and that ß-subunit myristoylation is important for transducing the metabolic stress signal.

Research and standardization in Alzheimer's trials: reaching international consensus.

Alzheimer's disease (AD) is an epidemic facing the entire world. Increased knowledge gained during the past 25 years indicates that AD falls along a clinical and neuropathological spectrum represented as a continuum that extends from preclinical disease in which there are no symptoms, through early symptomatic phases, and finally to AD dementia. The Alzheimer's research community recognizes that imaging, body fluids, and cognitive biomarkers contribute to enhanced diagnostic confidence for AD. There has also been emerging consensus regarding the use of AD biomarkers in clinical trials. The use of biomarkers in clinical trials and practice is hampered by the lack of standardization. In response to the emerging need for standardization, an international meeting of AD researchers was held in Melbourne, Australia, in March 2012 to bring together key researchers, clinicians, industry, and regulatory stakeholders with the aim of generating consensus on standardization and validation of cognitive, imaging, and fluid biomarkers, as well as lifestyle parameters used in research centers worldwide.

Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes.

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.

Fifteen Years of the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study: Progress and Observations from 2,359 Older Adults Spanning the Spectrum from Cognitive Normality to Alzheimer's Disease.

BACKGROUND: The Australian Imaging, Biomarkers and Lifestyle (AIBL) Study commenced in 2006 as a prospective study of 1,112 individuals (768 cognitively normal (CN), 133 with mild cognitive impairment (MCI), and 211 with Alzheimer's disease dementia (AD)) as an 'Inception cohort' who underwent detailed ssessments every 18 months. Over the past decade, an additional 1247 subjects have been added as an 'Enrichment cohort' (as of 10 April 2019). OBJECTIVE: Here we provide an overview of these Inception and Enrichment cohorts of more than 8,500 person-years of investigation. METHODS: Participants underwent reassessment every 18 months including comprehensive cognitive testing, neuroimaging (magnetic resonance imaging, MRI; positron emission tomography, PET), biofluid biomarkers and lifestyle evaluations. RESULTS: AIBL has made major contributions to the understanding of the natural history of AD, with cognitive and biological definitions of its three major stages: preclinical, prodromal and clinical. Early deployment of Aß-amyloid and tau molecular PET imaging and the development of more sensitive and specific blood tests have facilitated the assessment of genetic and environmental factors which affect age at onset and rates of progression. CONCLUSION: This fifteen-year study provides a large database of highly characterized individuals with longitudinal cognitive, imaging and lifestyle data and biofluid collections, to aid in the development of interventions to delay onset, prevent or treat AD. Harmonization with similar large longitudinal cohort studies is underway to further these aims.

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation.

The activation of AMP-activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl-CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser-221 phosphorylation does not always correlate with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside (AICAR) and contraction-stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle-specific kinase dead (KD) AMPK alpha2. In wild-type (WT) mice, AICAR and contraction increased AMPK alpha2 and alpha1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl-CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained, malonyl-CoA levels were reduced and rates of fatty acid oxidation were comparable between genotypes. During treadmill exercise both KD and WT mice had similar values of respiratory exchange ratio. These studies suggested the presence of an alternative ACC2 kinase(s). Using a phosphoproteomics-based approach we identified 18 Ser/Thr protein kinases whose phosphorylation was increased by greater than 25% in contracted KD relative to WT muscle. Utilizing bioinformatics we predicted that extracellular regulated protein-serine kinase (ERK1/2), inhibitor of nuclear factor (NF)-kappaB protein-serine kinase beta (IKKbeta) and protein kinase D (PKD) may phosphorylate ACC2 at Ser-221 but during in vitro phosphorylation assays only AMPK phosphorylated ACC2. These data demonstrate that AMPK is not essential for the regulation of fatty acid oxidation by AICAR or muscle contraction.

Mediterranean diet adherence and rate of cerebral Aß-amyloid accumulation: Data from the Australian Imaging, Biomarkers and Lifestyle Study of Ageing.

Accumulating research has linked Mediterranean diet (MeDi) adherence with slower cognitive decline and reduced Alzheimer's disease (AD) risk. However, no study to-date has examined the relationship between MeDi adherence and accumulation of cerebral Aß-amyloid (Aß; a pathological hallmark of AD) in older adults. Cognitively normal healthy control participants of the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study of Ageing completed the Cancer Council of Victoria Food Frequency Questionnaire at baseline, which was used to construct a MeDi score for each participant (score range 0-9; higher score indicating higher adherence). Cerebral Aß load was quantified by Pittsburgh Compound B positron emission tomography at baseline, 18 and 36 months: Only individuals categorised as "Aß accumulators", and thus considered to be on the AD pathway, were included in the analysis (N = 77). The relationship between MeDi adherence, MeDi components, and change in cerebral Aß load (baseline to 36 months) was evaluated using Generalised Linear Modelling, accounting for age, gender, education, Apolipoprotein E ε4 allele status, body mass index and total energy intake. Higher MeDi score was associated with less Aß accumulation in our cohort (ß = -0.01 ± 0.004, p = 0.0070). Of the individual MeDi score components, a high intake of fruit was associated with less accumulation of Aß (ß = -0.04 ± 0.01, p = 0.00036). Our results suggest MeDi adherence is associated with reduced cerebral AD pathology accumulation over time. When our results are considered collectively with previous data linking the MeDi to slower cognitive decline, it is apparent that MeDi adherence warrants further investigation in the quest to delay AD onset.

Associations of Dietary Protein and Fiber Intake with Brain and Blood Amyloid-ß.

Accumulating evidence suggests a diet high in protein and fiber may confer some protection against Alzheimer's disease (AD). However, no human studies to-date have assessed the relationship between protein and fiber intake, and plasma and brain amyloid-ß (Aß). Consequently, this cross-sectional study, investigated the association of self-reported dietary intakes of protein and fiber, with plasma and brain Aß burden (n = 541, and n = 162 respectively), in a well-characterized cohort of cognitively normal older adults, drawn from the larger Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging. We observed 12.59 and 8.43 higher odds of 'high' brain Aß burden (PiB PET SUVR≥1.5) if protein intake fell in the lowest and middle tertile, respectively, compared to the highest tertile (p = 0.008; p = 0.013). Thus, in this cohort, the more protein consumed, the less likelihood of 'high' Aß burden in the brain. No other significant associations were observed. The results of this study highlight the potentially protective impact of high dietary protein intake on brain Aß burden in older adults, before objective memory decline is apparent. While longitudinal validation is required, these findings may assist in the development of dietary approaches aimed at preventing or delaying AD onset.

Concordance Between Cerebrospinal Fluid Biomarkers with Alzheimer's Disease Pathology Between Three Independent Assay Platforms.

BACKGROUND: To enhance the accuracy of clinical diagnosis for Alzheimer's disease (AD), pre-mortem biomarkers have become increasingly important for diagnosis and for participant recruitment in disease-specific treatment trials. Cerebrospinal fluid (CSF) biomarkers provide a low-cost alternative to positron emission tomography (PET) imaging for in vivo quantification of different AD pathological hallmarks in the brains of affected subjects; however, consensus around the best platform, most informative biomarker and correlations across different methodologies are controversial. OBJECTIVE: Assessing levels of Aß-amyloid and tau species determined using three different versions of immunoassays, the current study explored the ability of CSF biomarkers to predict PET Aß-amyloid (32 Aß-amyloid-and 45 Aß-amyloid+), as well as concordance between CSF biomarker levels and PET Aß-amyloid imaging. METHODS: Prediction and concordance analyses were performed using a sub-cohort of 77 individuals (48 healthy controls, 15 with mild cognitive impairment, and 14 with AD) from the Australian Imaging Biomarker and Lifestyle study of aging. RESULTS: Across all three platforms, the T-tau/Aß42 ratio biomarker had modestly higher correlation with SUVR/BeCKeT (ρ= 0.69-0.8) as compared with Aß42 alone (ρ= 0.66-0.75). Differences in CSF biomarker levels between the PET Aß-amyloid-and Aß-amyloid+ groups were strongest for the Aß42/Aß40 and T-tau/Aß42 ratios (p < 0.0001); however, comparison of predictive models for PET Aß-amyloid showed no difference between Aß42 alone and the T-tau/Aß42 ratio. CONCLUSION: This study confirms strong concordance between CSF biomarkers and PET Aß-amyloid status is independent of immunoassay platform, supporting their utility as biomarkers in clinical practice for the diagnosis of AD and for participant enrichment in clinical trials.

A purine analog kinase inhibitor, calcium/calmodulin-dependent protein kinase II inhibitor 59, reveals a role for calcium/calmodulin-dependent protein kinase II in insulin-stimulated glucose transport.

Olomoucine is known as a cyclin-dependent kinase inhibitor. We found that olomoucine blocked insulin's ability to stimulate glucose transport. It did so without affecting the activity of known insulin signaling proteins. To identify the olomoucine-sensitive kinase(s), we prepared analogs that could be immobilized to an affinity resin to isolate binding proteins. One of the generated analogs inhibited insulin-stimulated glucose uptake with increased sensitivity compared with olomoucine. The IC(50) for inhibition of insulin-stimulated glucose uptake occurred at analog concentrations as low as 0.1 microM. To identify proteins binding to the analog, [(35)S]-labeled cell lysates prepared from 3T3-L1 adipocytes were incubated with analog chemically cross-linked to a resin support and binding proteins analyzed by SDS-PAGE. The major binding species was a doublet at 50-60 kDa, which was identified as calcium/calmodulin-dependent protein kinase II (CaMKII) by N-terminal peptide analysis and confirmed by matrix-assisted laser desorption ionization-mass spectrometry as the delta- and beta-like isoforms. To investigate CaMKII involvement in insulin-stimulated glucose uptake, 3T3-L1 adipocytes were infected with retrovirus encoding green fluorescent protein (GFP)-hemagluttinin tag (HA)-tagged CaMKII wild-type or the ATP binding mutant, K42M. GFP-HA-CaMKII K42M cells had less kinase activity than cells expressing wild-type GFP-HA-CaMKII. Insulin-stimulated glucose transport was significantly decreased (approximately 80%) in GFP-HA-CaMKII K42M cells, compared with nontransfected cells, and cells expressing either GFP-HA-CaMKII or GFP-HA. There was not a concomitant decrease in insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII K42M cells when compared with GFP-HA alone. However, insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII cells was significantly higher, compared with either GFP-HA or GFP-HA-CaMKII K42M cells. Our results implicate the involvement of CaMKII in glucose transport in a permissive role.

Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase.

Although interleukin-6 (IL-6) has been associated with insulin resistance, little is known regarding the effects of IL-6 on insulin sensitivity in humans in vivo. Here, we show that IL-6 infusion increases glucose disposal without affecting the complete suppression of endogenous glucose production during a hyperinsulinemic-euglycemic clamp in healthy humans. Because skeletal muscle accounts for most of the insulin-stimulated glucose disposal in vivo, we examined the mechanism(s) by which IL-6 may affect muscle metabolism using L6 myotubes. IL-6 treatment increased fatty acid oxidation, basal and insulin-stimulated glucose uptake, and translocation of GLUT4 to the plasma membrane. Furthermore, IL-6 rapidly and markedly increased AMP-activated protein kinase (AMPK). To determine whether the activation of AMPK mediated cellular metabolic events, we conducted experiments using L6 myotubes infected with dominant-negative AMPK alpha-subunit. The effects described above were abrogated in AMPK dominant-negative-infected cells. Our results demonstrate that acute IL-6 treatment enhances insulin-stimulated glucose disposal in humans in vivo, while the effects of IL-6 on glucose and fatty acid metabolism in vitro appear to be mediated by AMPK.

Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase.

Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.

Modulation of Retinal Arteriolar Central Reflection by APOE Genotype.

OBJECTIVE: This study investigated the retinal arteriolar central reflex (CR, the central reflection observed in photographs of retinal vessels), which may provide information about micro-vascular health in the retina and also the brain, due to the homology between these vascular networks. The study also describes a novel computer based semi-automated technique that accurately quantifies retinal arteriolar CR and vessel width, and calculates the CR to vessel width ratio (CRR) from digital retinal photographs. METHODS: Digital retinal photographs were collected from participants in the Australian Imaging, Biomarkers and Lifestyle study of ageing (AIBL), including 25 participants diagnosed with Alzheimer's disease (AD) (age 72.4 ± 7.5 yrs, 12 male, 13 female) and 123 elderly participants without dementia (cognitively normals: CN) (age 71.6 ± 5.6 yrs, 55 male, 68 female). Using a sub-cohort of 144 (22 AD, 122 CN) with the novel CRR measures, we identified significantly higher CRR levels in AD participants (mean CRR 0.253 (SD 0.04)) as compared with CN's (mean CRR 0.231 (SD 0.04), p = 0.025). Adjustment for APOE ε4 allele status however, reduced the significance (p = 0.081). CRR was significantly higher in APOE ε4 allele carriers (mean CRR 0.254 (SD 0.03) as compared with non-carriers (mean CRR 0.224 (SD 0.05), p < 0.0001). RESULTS: These data indicate that CRR is strongly linked to APOE ε4 status and exhibits a weaker, independent trend with AD diagnosis. The retina may be useful as a novel model for non-invasive monitoring of the effects of APOE ε4 on the central nervous system, particularly in cerebrovascular disease.