An accessible pharmacodynamic transcriptional biomarker for notch target engagement.

γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

Short-term metabolic effects of prednisone administration in healthy subjects.

AIMS: Supraphysiologic glucocorticoid activity is well established to cause impaired glucose tolerance and insulin resistance, yet no study has evaluated dose-dependent effects of low-dose prednisone during short-term oral administration. METHODS: The objective of this study was to quantify the effects of daily 10 or 25 mg prednisone administration for one week on insulin sensitivity by employing a two-step hyperinsulinemic euglycemic glucose clamp (Step 1: insulin infusion = 20 mU/m²/min; Step 2: insulin infusion = 80 mU/m²/min) in healthy, lean males. The amount of glucose infused at steady-state to maintain stable blood glucose [90 mg/dl (4.95 mmol/l)] was used to calculate several indices of insulin sensitivity. RESULTS: During Step 1 of the clamp, whole body glucose disposal (M) was reduced by 35% (p = 0.003) and M/I was reduced by 29% (p = 0.025) for 25 mg prednisone compared to placebo. No appreciable effect of 10 mg prednisone was observed. During Step 2, M was reduced by 33% (p = 0.001) and 15% (p = 0.006) for 25 and 10 mg prednisone compared to placebo; and M/I ratio was reduced by 31% (p < 0.001) and 13% (p = 0.026), respectively. The insulin sensitivity index, Si, calculated as the quotient of augmentation of M/I between Step 1 and 2, was reduced by 35.3% (p < 0.01) and 23.5% (p < 0.05) for 25 and 10 mg prednisone, respectively. CONCLUSION: Administration of relatively low pharmacological doses of prednisone for one week impaired insulin sensitivity in a dose-dependent manner in healthy males. These observed changes in insulin sensitivity are likely to be clinically relevant, especially in individuals predisposed to develop glucose intolerance.

Potent and selective agonism of the melanocortin receptor 4 with MK-0493 does not induce weight loss in obese human subjects: energy intake predicts lack of weight loss efficacy.

MK-0493 is a novel, potent, and selective agonist of the melanocortin receptor 4 (MC4R), one of the best-validated genetic targets and considered one of the most promising for the development of antiobesity therapeutics. An ad libitum energy-intake model was qualified with excellent reproducibility: the geometric mean ratio (GMR) with 95% confidence interval (CI) for total energy intake over a period of 24 h for 30 mg sibutramine/placebo was 0.82 (0.76, 0.88), and for 10 mg sibutramine/placebo it was 0.98 (0.91, 1.05). MK-0493 showed a small and marginally significant effect on 24-h energy intake, whereas 30 mg of sibutramine caused a significant reduction in total 24-h energy intake; specifically, the GMR (95% CI) for 30 mg sibutramine/placebo was 0.79 (0.74, 0.85). MK-0493 was associated with modest weight reduction from baseline but had only small, statistically insignificant effects relative to placebo after 12 weeks in a fixed-dose study and also after 18 weeks of stepped-titration dosing. We conclude that agonism of MC4R is not likely to represent a viable approach to the development of antiobesity therapeutics.

Dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes: focus on sitagliptin.

Dipeptidyl peptidase-4 (DPP-4) inhibitors represent a new class of oral antihyperglycemic agents to treat patients with type 2 diabetes. DPP-4 inhibitors improve fasting and postprandial glycemic control without hypoglycemia or weight gain. This article focuses on the physiology, clinical pharmacology, tolerability, and clinical utility of the DPP-4 inhibitor sitagliptin in the management of type 2 diabetes.

Identification of discrete classes of endosome-derived small vesicles as a major cellular pool for recycling membrane proteins.

Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5'-[gamma-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.

GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4.

Some cell types have cytoplasmic storage vesicles whose fusion with the cell surface is triggered by an extracellular signal. To explore the relationship between different classes of storage vesicles, we expressed, in the neuro-endocrine cell line PC12, the facilitative glucose transporter GLUT4, which is stored in small cytoplasmic vesicles in fat and muscle cells and mobilized to the cell surface when insulin is present. PC12 cells have two known types of storage vesicles, secretory granules and synaptic vesicles, but GLUT4 is targeted to neither. It is recovered, however, in a class of small vesicles that sediment approximately twice as fast as synaptic vesicles. Immunoelectron microscopy confirmed the presence of such small vesicles in transfected PC12 cells. By velocity sedimentation analysis, GLUT4 vesicles efficiently exclude the synaptic vesicle markers synaptophysin, SV2, and synaptobrevin; the transferrin receptor, a marker of conventional endocytosis; and the polymeric immunoglobulin receptor, a marker of transcytosis. The exclusion of synaptophysin and the transferrin receptor from most of the GLUT4-containing structures was confirmed by confocal immunofluorescence microscopy. Like synaptic vesicles, therefore, GLUT4 vesicles of PC12 cells appear to be a unique type of organelle. A GLUT4-containing organelle of identical sedimentation properties was found in transfected fibroblast cell lines and in rat adipocytes. On stimulation of the adipocytes with insulin, GLUT4 was translocated from the peak of small vesicles to faster sedimenting membranes. We propose that the class of vesicles described here is present in a wide range of cell types and is involved in transient modification of the cell surface.

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells.

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207-1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle-like structures as well as from secretory granules.

The human mannose-binding protein gene. Exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10.

The human mannose-binding protein (MBP) plays a role in first line host defense against certain pathogens. It is an acute phase protein that exists in serum as a multimer of a 32-kD subunit. The NH2 terminus is rich in cysteines that mediate interchain disulphide bonds and stabilize the second collagen-like region. This is followed by a short intervening region, and the carbohydrate recognition domain is found in the COOH-terminal region. Analysis of the human MBP gene reveals that the coding region is interrupted by three introns, and all four exons appear to encode a distinct domain of the protein. It appears that the human MBP gene has evolved by recombination of an ancestral nonfibrillar collagen gene with a gene that encodes carbohydrate recognition, and is therefore similar to the human surfactant SP-A gene and the rat MBP gene. The gene for MBP is located on the long arm of chromosome 10 at 10q11.2-q21, a region that is included in the assignment for the gene for multiple endocrine neoplasia type 2A.

A human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins.

Mannose-binding proteins have been isolated from the liver of rats and humans and subsequently been found in the serum of rats, rabbits, and humans. We report the isolation of cDNA clones isolated from a human liver cDNA library that encodes a human mannose-binding protein. The primary structure has three domains: (a) an NH2-terminal cysteine-rich segment of 19 amino acids which appears to be involved in the formation of interchain disulfide bonds that would stabilize multimeric forms of the protein; (b) a collagen-like region consisting of 19 repeats of the sequence Gly-x-y; and (c) a COOH-terminal putative carbohydrate-binding domain consisting of 148 residues. This human mannose-binding protein bears 51% overall homology (allowing three gaps) with a rat mannose-binding protein C and 48% homology (allowing seven gaps) with a rat mannose-binding protein A. Like these homologous rat proteins, the human mannose-binding protein COOH-terminal sequences are homologous to the carbohydrate recognition portion of several other lectin-like proteins including mammalian hepatic receptors, an insect-soluble hemolymph, and a sea urchin lectin found in coelomic fluid. The apoproteins of dog and human surfactant and the human lymphocyte IgE Ec receptor have not been shown to have lectin-like properties, yet by homology are members of this family of lectin-like proteins. The human mannose-binding protein is preceded by a typical hydrophobic signal sequence and its hepatic secretion is induced as part of the acute-phase response consistent with its probable role in host defense.