OBJECTIVE Insulin resistance is a powerful risk factor for Type 2 diabetes and a constellation of chronic diseases, and is most commonly associated with obesity. We examined if factors other than obesity are more substantial predictors of insulin sensitivity under baseline, non-stimulated conditions. DESIGN AND METHODS Metabolic assessment was performed in healthy dogs (n=90). Whole-body sensitivity from euglycemic clamps (SICLAMP) was the primary outcome variable, and was measured independently by IVGTT (n=36). Adiposity was measured by MRI (n=90), and glucose-stimulated insulin response was measured from hyperglycemic clamp or IVGTT (n=86 and 36, respectively). RESULTS SICLAMP was highly variable (5.9 to 75.9 dl/min per kg per μU/ml). Despite narrow range of body weight (mean, 28.7±0.3 kg), adiposity varied ∼8-fold and was inversely correlated with SICLAMP (p<0.025). SICLAMP was negatively associated with fasting insulin, but most strongly associated with insulin clearance. Clearance was the dominant factor associated with sensitivity (r=0.53, p<0.00001), whether calculated from clamp or IVGTT. CONCLUSIONS These data suggest that insulin clearance contributes substantially to insulin sensitivity, and may be pivotal in understanding the pathogenesis of insulin resistance. We propose that hyperinsulinemia due to reduction in insulin clearance is responsible for insulin resistance secondary to changes in body weight.

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

Recent data suggests that metallothioneins (MTs) are major neuroprotective proteins within the CNS. In this regard, we have recently demonstrated that MT-IIA (the major human MT-I/-II isoform) promotes neural recovery following focal cortical brain injury. To further investigate the role of MTs in cortical brain injury, MT-I/-II expression was examined in several different experimental models of cortical neuron injury. While MT-I/-II immunoreactivity was not detectable in the uninjured rat neocortex, by 4 days, following a focal cortical brain injury, MT-I/-II was found in astrocytes aligned along the injury site. At latter time points, astrocytes, at a distance up to several hundred microns from the original injury tract, were MT-I/-II immunoreactive. Induced MT-I/-II was found both within the cell body and processes. Using a cortical neuron/astrocyte co-culture model, we observed a similar MT-I/-II response following in vitro injury. Intriguingly, scratch wound injury in pure astrocyte cultures resulted in no change in MT-I/-II expression. This suggests that MT induction was specifically elicited by neuronal injury. Based upon recent reports indicating that MT-I/-II are major neuroprotective proteins within the brain, our results provide further evidence that MT-I/-II plays an important role in the cellular response to neuronal injury. Keywords: brain injury, metallothionein, neuronal injury. While MTs are often considered in the context of zinc metabolism, or protection from free radical damage (see commentary by Palmiter 1998), more recently there have been reports indicating that this protein confers a protective effect following brain injury. Indeed, MT-I and MT-II knockout mice are susceptible to physical, chemical and ischemic brain injury (Penkowa et al. 1999a(Penkowa et al. , 1999bCarrasco et al. 2000;Trendelenburg et al. 2002), while mice that overexpress MT isoforms in the brain are comparatively more resistant to injury (Campagne et al. 1999;Giralt et al. 2002;Penkowa et al. 2002).The mechanism by which MT is protective is currently unknown. Possibilities include the zinc-binding properties of the protein (seven zinc ions per protein molecule) or its ability to scavenge free radicals. An intriguing possibility, which has recently arisen from the work of Hidalgo and colleagues, is that MT is able to reduce inflammation associated with CNS injury, leading to enhanced recovery Penkowa and Hidalgo 2001). Interestingly, we have recently discovered that human MT-IIA has a previously unsuspected ability to enhance neuronal Received April 6, 2003; revised manuscript received September 29, 2003; accepted September 29, 2003. Address correspondence and reprint requests to Roger S. Chung, NeuroRepair Group, School of Medicine, University of Tasmania, PO Box 252-58, Hobart, Tasmania 7001, Australia. E-mail: rschung@utas.edu.auAbbreviations used: FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; MT, metallothionein; MT-I/-II, metallothionein-I and -II; PBS, phosphate-buffered saline; PI, po...

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BackgroundAxon degeneration, a key pathological event in many neurodegenerative diseases and injury, can be induced by somatodendritic excitotoxin exposure. It is currently unclear, however, whether excitotoxin-induced axon degeneration is mechanistically similar to Wallerian degeneration, which occurs following axon transection, but does not involve axonal caspase activation.ResultsWe have used mouse primary cortical neurons at 9 days in vitro, in a compartmented culture model that allows separation of the axon from the soma, to examine the pathological cascade of excitotoxin-induced axon degeneration. Excitotoxicity induced by chronic exposure to kainic acid, resulted in axonal fragmentation, which was associated with activation of caspase-3 in the axonal compartment. To examine the role of microtubules in these events, the microtubule-stabilizing agent, taxol, was added to either the axonal or somatodendritic compartment. Our results demonstrated that microtubule stabilization of axons resulted in a significant reduction in the number of fragmented axons following excitotoxin exposure. Interestingly, taxol exposure to either the somatodendritic or axonal compartment resulted in reduced caspase-3 activation in axons, suggesting that caspase activation is a downstream event of microtubule destabilization and involves signalling from the cell soma.ConclusionThese data suggest that excitotoxin-induced axon degeneration shows some mechanistic differences to Wallerian degeneration, and that microtubule stabilization may assist in protecting nerve cells from excitotoxic effects.

Early-life cognitive enrichment may reduce the risk of experiencing cognitive deterioration and dementia in later-life. However, an intervention to prevent or delay dementia is likely to be taken up in mid to later-life. Hence, we investigated the effects of environmental enrichment in wildtype mice and in a mouse model of Aβ neuropathology (APP /PS1 ) from 6 months of age. After 6 months of housing in standard laboratory cages, APP /PS1 (n = 27) and healthy wildtype (n = 21) mice were randomly assigned to either enriched or standard housing. At 12 months of age, wildtype mice showed altered synaptic protein levels and relatively superior cognitive performance afforded by environmental enrichment. Environmental enrichment was not associated with alterations to Aβ plaque pathology in the neocortex or hippocampus of APP /PS1 mice. However, a significant increase in synaptophysin immunolabeled puncta in the hippocampal subregion, CA1, in APP /PS1 mice was detected, with no significant synaptic density changes observed in CA3, or the Fr2 region of the prefrontal cortex. Moreover, a significant increase in hippocampal BDNF was detected in APP /PS1 mice exposed to EE, however, no changes were detected in neocortex or between Wt animals. These results demonstrate that mid to later-life cognitive enrichment has the potential to promote synaptic and cognitive health in ageing, and to enhance compensatory capacity for synaptic connectivity in pathological ageing associated with Aβ deposition.

In recent years metallothionein (MT) biology has moved from investigation of its ability to protect against environmental heavy metals to a wider appreciation of its role in responding to cellular stress, whether as a consequence of normal function, or following injury and disease. This is exemplified by recent investigation of MT in the mammalian brain where plausible roles for MT action have been described, including zinc metabolism, free radical scavenging, and protection and regeneration following neurological injury. Along with other laboratories we have used several models of central nervous system (CNS) injury to investigate possible parallels between injury-dependent changes in MT expression and those observed in the ageing and/or degenerating brain. Therefore, this brief review aims to summarise existing information on MT expression during CNS ageing, and to examine the possible involvement of this protein in the course of human neurodegenerative disease, as exemplified by Alzheimer's disease.

Disruption of leptin signalling has been implicated as playing a role in the development of Alzheimer's disease (AD). Leptin has previously been shown to be affected by amyloid-beta (Aβ)-related signalling; however, pathways that link leptin to the disease pathogenesis have not been determined. To characterize the association between increasing age-dependent Aβ levels with leptin signalling and the vulnerable brain regions in AD, we assessed the mRNA and protein expression profile of leptin and leptin receptor (Ob-Rb) at 9 and 18-month-age in APP/PS1 mice. Immunohistochemical labelling demonstrated that leptin and Ob-Rb proteins were localised to neocortical and hippocampal neurons in APP/PS1 and wildtype (WT) mice. Neuronal leptin and Ob-Rb immunolabelling was more prominent in the neocortex of both groups at 9 month of age, while, at 18 months, labelling was reduced in the hippocampus of APP/PS1 mice relative to WT. Immunoblotting analysis demonstrated decreased hippocampal leptin levels, concomitantly with an increased Ob-Rb levels, in APP/PS1 mice compared with WT controls at 18 month of age. While no leptin mRNA was found in either of the groups analysed, Ob-Rb mRNA was significantly decreased in the hippocampus of APP/PS1 mice at both ages analysed. In addition, a significant decreased protein kinase B (Akt) activity concomitantly with an upregulation of suppressor of cytokine signaling-3 (SOCS3) and protein-tyrosine phosphatase 1B (PTP1B) transcripts was present. Thus, these results collectively indicate alterations of leptin signalling in the hippocampus of APP/PS1 mice, providing novel insights about the pathways that could link aberrant leptin signaling to the pathological changes of AD.