Background and Aim: Exenatide is a glucagon-like peptide-1 receptor agonist demonstrated to improve glycemic control with low hypoglycemia risk in patients with type 2 diabetes mellitus. The Diabetes Therapy Utilization: Researching Changes in A1C, Weight, and Other Factors Through Intervention With Exenatide Once Weekly (DURATION) program comprised 6 randomized, comparator-controlled, 24- to 30-week trials of exenatide once weekly (EQW), an extended-release formulation. This post hoc analysis pooled data from patients taking EQW across 6 trials to assess efficacy and safety in a large, varied patient population. Materials and Methods: The intent-to-treat (ITT) population contained 1379 patients (baseline mean + standard deviation glycated hemoglobin [HbA(1c)] levels of 8.4% +/- 1.1%) who were treated with EQW over the course of 24 to 30 weeks. Changes from baseline in efficacy parameters for the ITT population and a completer population (1195 patients with >= 22 weeks of exposure) were evaluated. Results: The ITT population experienced significant reductions from baseline (least-squares mean [95% CI]) in HbA(1c) levels (-1.4% [-1.5% to -1.4%]), fasting blood glucose levels (-36 mg/dL [-38.4 mg/dL to -33.8 mg/dL]), and body weight (-2.5 kg [-2.8 kg to -2.3 kg]) after 24 to 30 weeks of EQW treatment. Reductions in HbA(1c) and fasting blood glucose levels were observed across baseline HbA(1c) level strata; patients with higher baseline HbA(1c) levels experienced greater reductions. Treatment with EQW was associated with modest, significant reductions in blood pressure (systolic blood pressure, -2.8 mm Hg [-3.5 mm Hg to -2.1 mm Hg]; diastolic blood pressure, -0.8 mm Hg [-1.2 mm Hg to -0.4 mm Hg]), and fasting lipid levels (total cholesterol, -6.5 mg/dL [-8.2 mg/dL to -4.7 mg/dL]; low-density lipoprotein cholesterol, -3.9 mg/dL [5.3 mg/dL to -2.5 mg/dL]; and triglyceride [geometric least-squares mean percent change (95% CI)], -6% [-8% to -4%] levels). Similar reductions were observed in the completer population. Exenatide once weekly was generally well tolerated. Transient, mild-to-moderate gastrointestinal treatment-emergent adverse events and injection-site treatment-emergent adverse events were reported most frequently, but were seldom treatment limiting. No major hypoglycemic events were observed; minor hypoglycemic events occurred infrequently in patients not using a sulfonylurea. Conclusion: This post hoc analysis of > 1300 patients demonstrated that EQW was associated with significant reductions in HbA(1c) levels, body weight, blood pressure, and fasting lipid levels, with minimal hypoglycemia risk. Consistent with established safety profiles, EQW therapy was generally well tolerated.

Neurons rely on microtubule (MT) motor proteins such as kinesin‐1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK‐3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin‐1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK‐3 is a required negative regulator of both kinesin‐1‐mediated and dynein‐mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK‐3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK‐3 regulates transport by altering the activity of kinesin‐1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK‐3 and APP, and demonstrate that endogenous GSK‐3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK‐3 controls the number of active motors that are moving a cargo.

Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain.

Neurons rely on microtubule motor proteins such as kinesin-1 and dynein to transport essential cargos along the axon. Defective transport is connected to neurodegenerative diseases, including Alzheimer's disease. Glycogen Synthase Kinase 3(GSK-3) has been proposed to be a central player in Alzheimer's. We show that GSK-3 is a required negative regulator of both kinesin-1-mediated and dynein-mediated transport of the Amyloid Precursor Protein, a key contributor to Alzheimer's pathology. By measuring the forces motors generate in vivo, we find that GSK-3 regulates transport by altering the activity of kinesin-1 motors but not their binding to the cargo leading us to propose that this regulation occurs via a mechanism of changing motor-microtubule interactions.

AimsType 2 diabetes mellitus (T2DM) is often associated with cardiovascular (CV) risk factors such as obesity, hypertension and dyslipidemia. The objective of this analysis was to evaluate potential effects of exenatide once weekly (ExQW), a GLP‐1 receptor agonist, on glycaemic control and CV risk factors.MethodsThis analysis included 675 Intent‐to‐Treat patients with T2DM [baseline (mean ± SD) HbA1c, 8.1 ± 1.2%; fasting blood glucose (FBG), 166 ± 48 mg/dl; weight, 94.3 ± 19.4 kg; systolic/diastolic blood pressure (SBP/DBP), 129 ± 15/78 ± 9 mm Hg; total cholesterol, 178.5 ± 41.9 mg/dl; low‐density lipoprotein (LDL), 100.1 ± 35.0 mg/dl; high‐density lipoprotein (HDL), 44.5 ± 11.6 mg/dl; triglycerides, 155.6 ± 3.3 mg/dl; alanine aminotransferase (ALT), 32.1 ± 19.5 U/l] treated with diet and exercise alone or in combination with metformin, sulfonylurea, and/or thiazolidinedione who received 52 weeks of ExQW in four clinical trials.ResultsAt 52 weeks, ExQW significantly improved HbA1c [mean (SE) change from baseline, −1.3 (0.05)%], FBG [−36.3 (2.02) mg/dl], body weight [−2.6 (0.19) kg], SBP/DBP [−3.6 (0.56) mm Hg/−1.2 (0.34) mm Hg], total cholesterol, −4.4 (1.33) mg/dl; LDL, −2.6 (1.08) mg/dl; HDL, 1.1 (0.31) mg/dl; triglycerides, −7 (1.6)%], and ALT [−4.3 (0.71) IU/l] concentrations, with greater improvements in patients with elevated analyte levels at baseline. Improvements were observed across a range of background antihyperglycaemia therapies. Of patients completing 52 weeks, 19% achieved the composite American Diabetes Association goal (HbA1c < 7.0%, BP < 130/80 mm Hg, LDL < 100 mg/dl), compared to 1% at baseline. Nearly half (48%) achieved HbA1c < 7.0% without weight gain or major/minor hypoglycaemia. Nausea was the most frequent adverse event and was predominantly mild. Hypoglycaemia was infrequent, and more common with a sulfonylurea.ConclusionsWith 52 weeks of ExQW, patients experienced sustained improvements in glycaemic control and CV risk factors, with an increased likelihood of achieving both a clinically relevant composite outcome (HbA1c < 7% without weight gain or increased risk of hypoglycaemia) and a composite of key therapeutic goals (HbA1c < 7%, BP < 130/80 mm Hg, LDL < 100 mg/dl).

Abnormal deposits of amyloid plaques which characterize Alzheimer's disease have been linked to deficient transport of the Amyloid Precursor Protein (APP) by kinesin-1 motors. It is of great interest to identify the regulatory factors that enhance or inhibit axonal transport, thereby providing direction along the path of pharmaceutical treatments for neurodegenerative diseases. The serine/threonine kinase Glycogen Synthase Kinase 3 (GSK-3) has been proposed as a regulator of axonal kinesin-1 and has been linked to many aspects of the development of Alzheimer's disease.

A long-standing question in developmental biology is how amphibians establish a dorsoventral axis. The prevailing view has been that cortical rotation is used to move a dorsalizing activity from the bottom of the egg towards the future dorsal side. We review recent evidence that kinesin-dependent movement of particles containing components of the Wnt intracellular pathway contributes to the formation of the dorsal organizer,and suggest that cortical rotation functions to align and orient microtubules,thereby establishing the direction of particle transport. We propose a new model in which active particle transport and cortical rotation cooperate to generate a robust movement of dorsal determinants towards the future dorsal side of the embryo.

In Xenopus, axis development is initiated by dorsally elevated levels of cytoplasmic β-catenin, an intracellular factor regulated by GSK3 kinase activity. Upon fertilization, factors that increase β-catenin stability are translocated to the prospective dorsal side of the embryo in a microtubule-dependent process. However, neither the identity of these factors nor the mechanism of their movement is understood. Here, we show that the GSK3 inhibitory protein GBP/Frat binds kinesin light chain (KLC), a component of the microtubule motor kinesin. Upon egg activation, GBP-GFP and KLC-GFP form particles and exhibit directed translocation. KLC, through a previously uncharacterized conserved domain, binds a region of GBP that is required for GBP translocation and for GSK3 binding, and competes with GSK3 for GBP. We propose a model in which conventional kinesin transports a GBP-containing complex to the future dorsal side, where GBP dissociates and contributes to the local stabilization of β-catenin by binding and inhibiting GSK3.

This chapter discusses the automatic eukaryotic artificial chromosomes (AEAC). Evolutionary theory proposes that mitochondria, plastids, and chloroplasts originated by the engulfment or cell fusion of prokaryotes by eukaryotes. As this endo-symbiotic relationship evolved, the size of the bacterial DNA genome decreased and the functions of genes lost from the bacterial genome were assumed by the eukaryotic chromosome. These experiments demonstrate a new technology that could be used for transferring an entire prokaryotic genome or other large DNA molecules, in circular form, into a eukaryotic cell, where the circular DNA is automatically converted into an artificial linear chromosome in vivo. The production of AEAC-bacterial genomes for animals, human cells (AHACs), and plants preferably require different telomeres, centromeres, and selectable markers that function in these cells to make automatic chromosomes. AHACs carrying genomic or cDNAs for the necessary genes to be transferred would be ideal because of their formation of automatic functional chromosomes upon reaching the nuclei of the human cells.

The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear β-catenin/Tcf complex, activating Wnt target genes. A crystal structure of β-catenin bound to the β-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to β-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in β-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is β-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal–ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and β-catenin form a complex that promotes the GSK-3–mediated phosphorylation and subsequent degradation of β-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates β-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3–mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic β-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in β-catenin levels and establishment of the dorsal axis.

Wnt proteins transduce their signals through dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3β (GSK), leading to the accumulation of cytosolic β‐catenin and activation of TCF/LEF‐1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF‐1, we investigated the roles of Axin and Frat1 in Wnt‐mediated activation of LEF‐1 in mammalian cells. We found that Dvl interacts with Axin and with Frat1, both of which interact with GSK. Similarly, the Frat1 homolog GBP binds Xenopus Dishevelled in an interaction that requires GSK. We also found that Dvl, Axin and GSK can form a ternary complex bridged by Axin, and that Frat1 can be recruited into this complex probably by Dvl. The observation that the Dvl‐binding domain of either Frat1 or Axin was able to inhibit Wnt‐1‐induced LEF‐1 activation suggests that the interactions between Dvl and Axin and between Dvl and Frat may be important for this signaling pathway. Furthermore, Wnt‐1 appeared to promote the disintegration of the Frat1–Dvl–GSK–Axin complex, resulting in the dissociation of GSK from Axin. Thus, formation of the quaternary complex may be an important step in Wnt signaling, by which Dvl recruits Frat1, leading to Frat1‐mediated dissociation of GSK from Axin.