Type 2 diabetes (T2D), which has currently become a global pandemic, is a metabolic disease largely characterised by impaired insulin secretion and action. Significant progress has been made in understanding T2D aetiology and pathogenesis, which is discussed in this review. Extrapancreatic pathology is also summarised, which demonstrates the highly multifactorial nature of T2D. Glucagon-like peptide (GLP)-1 is an incretin hormone responsible for augmenting insulin secretion from pancreatic beta-cells during the postprandial period. Given that native GLP-1 has a very short half-life, GLP-1 mimetics with a much longer half-life have been developed, which are currently an effective treatment option for T2D by enhancing insulin secretion in patients. Interestingly, there is continual emerging evidence that these therapies alleviate some of the post-diagnosis complications of T2D. Additionally, these therapies have been shown to induce weight loss in patients, suggesting they could be an alternative to bariatric surgery, a procedure associated with numerous complications. Current GLP-1-based therapies all act as orthosteric agonists for the GLP-1 receptor (GLP-1R). Interestingly, it has emerged that GLP-1R also has allosteric binding sites and agonists have been developed for these sites to test their therapeutic potential. Recent studies have also demonstrated the potential of bi- and tri-agonists, which target multiple hormonal receptors including GLP-1R, to more effectively treat T2D. Improved understanding of T2D aetiology/pathogenesis, coupled with the further elucidation of both GLP-1 activity/targets and GLP-1R mechanisms of activation via different agonists, will likely provide better insight into the therapeutic potential of GLP-1-based therapies to treat T2D.

Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.

Clustering of inhibitory γ‐aminobutyric acid_A (GABA_A) and glycine receptors at synapses is thought to involve key interactions between the receptors, a “scaffolding” protein known as gephyrin and the RhoGEF collybistin. We report the identification of a balanced chromosomal translocation in a female patient presenting with a disturbed sleep‐wake cycle, late‐onset epileptic seizures, increased anxiety, aggressive behavior, and mental retardation, but not hyperekplexia. Fine mapping of the breakpoint indicates disruption of the collybistin gene (ARHGEF9) on chromosome Xq11, while the other breakpoint lies in a region of 18q11 that lacks any known or predicted genes. We show that defective collybistin transcripts are synthesized and exons 7–10 are replaced by cryptic exons from chromosomes X and 18. These mRNAs no longer encode the pleckstrin homology (PH) domain of collybistin, which we now show binds phosphatidylinositol‐3‐phosphate (PI3P/PtdIns‐3‐P), a phosphoinositide with an emerging role in membrane trafficking and signal transduction, rather than phosphatidylinositol 3,4,5‐trisphosphate (PIP3/PtdIns‐3,4,5‐P) as previously suggested in the “membrane activation model” of gephyrin clustering. Consistent with this finding, expression of truncated collybistin proteins in cultured neurons interferes with synaptic localization of endogenous gephyrin and GABA_A receptors. These results suggest that collybistin has a key role in membrane trafficking of gephyrin and selected GABA_A receptor subtypes involved in epilepsy, anxiety, aggression, insomnia, and learning and memory. Hum Mutat 0,1–9, 2008.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃]. In order to study the effects of PtdIns(3,4,5)P₃ on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P₃, inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P₄], with high affinity (K_d 32.2±5.2 nM) and inositol phosphate specificity [K_d values for Ins(1,3,4,5,6)P₅, InsP₆, Ins(1,3,4)P₃ and Ins(1,4,5)P₃: 283±32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P₄, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P₃ receptor. To address whether GRP1 binds PtdIns(3,4,5)P₃in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP–GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P₃. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 µM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P₃ and hence constitutes a true PtdIns(3,4,5)P₃ receptor.

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.