Diabetes affects over 16 million Americans yearly, resulting in hyperglycaemia and microvascular complications, including retinopathy, neuropathy and nephropathy. Animal models have been developed to examine the immunological aspects of type 1 diabetes and the pathogenic mechanisms associated with diabetic retinopathy, but the methods of diabetes induction raise concerns regarding these models. Zebrafish (Danio rerio) have been used extensively to study developmental processes and mutant zebrafish strains have been used to examine vision disease present in humans. In this paper, we have induced hyperglycaemia in zebrafish by alternately immersing the fish in glucose solution or water. Eyes from untreated fish or fish exposed to alternating glucose/water solutions for 28 days were dissected, sectioned and stained to visualise cell bodies in the retina. In untreated fish retinas, the inner plexiform layer (IPL) and inner nuclear layer (INL) were approximately the same thickness, whereas in fish repeatedly exposed to glucose solutions the IPL was approximately 55% the thickness of the INL. Both the IPL and INL were significantly reduced in retinas of treated fish, compared to untreated fish, similar to that seen in other animal models of diabetes and in diabetic patients. These results suggest that zebrafish may be used as an animal model in which to study diabetic retinopathy.

Stable-isotope signatures in animal tissues presumably reflect the local food web. However, that assumption may be complicated by differential nutrient routing, fractionation, and the possibility that large organisms are not in isotopic equilibrium with seasonally available food sources. Additionally, the rate at which organisms incorporate the isotopic signature of a food is largely unknown. In this study we assessed the rate of carbon- and nitrogen-isotope turnover in liver, muscle, and blood in mice (Mus musculus L., 1758) following a diet change. We report the proportion of tissue turnover caused by growth versus that caused by metabolic tissue replacement. Growth accounted for approximately 10% of observed tissue turnover in adult mice. Blood carbon had the shortest half-life (16.9 days), followed by muscle carbon (23.9 days). Liver carbon turnover, which was slower than blood and muscle carbon turnovers, was not as well described by the exponential decay equations. All tissues primarily reflect the protein carbon signature rather than the carbohydrate carbon signature. The nitrogen signature in all tissues was enriched by 3‰–5‰ over their diets' nitrogen signature, depending on tissue type, and the isotopic turnover rates of nitrogen in blood and muscle were comparable with those observed for carbon.

Stable isotopes have proven to be a useful tool for deciphering food webs, examining migration patterns and determining nutrient resource allocation. In order to increase the descriptive power of isotopes, an increasing number of studies are using them to model tissue turnover. However, these studies have, mostly by necessity, been largely limited to laboratory experiments and the demand for an easier method of estimating tissue turnover in the field for a large variety of organisms remains. In this study, we have determined the turnover rate of blood in mice and rats using stable isotope analysis, and compared these rates to the metabolic rates of the animals. Rats (Rattus norvegicus) (n=4) and mice (Mus musculus) (n=4) were switched between isotopically distinct diets, and the rate of change of delta(13)C and delta(15)N in whole blood was determined. Basal metabolic rates (as CO(2) output and O(2) consumption per unit time, normalized for mass) were determined for the rats and mice. Rats, which were an order of magnitude larger and had a slower metabolic rate per unit mass than mice (0.02 vs. 0.14 O(2)/min/g), had a slower blood turnover than mice for (13)C (t (1/2 )=24.8 and 17.3 days, respectively) and (15)N (t (1/2 )=27.7 and 15.4 days, respectively). A positive correlation between metabolic rate and blood isotopic turnover rate was found. These are the only such data for mammals available, but the literature for birds shows that mass and whole-body metabolic rates in birds scale logarithmically with tissue turnover. Interestingly, the mammalian data graph separately from the bird data on a turnover versus metabolic rate plot. Both mice and rat tissue in this study exhibited a slower turnover rate compared to metabolic rate than for birds. These data suggest that metabolic rate may be used to estimate tissue turnover rate when working with organisms in the field, but that a different relationship between tissue turnover and metabolism may exist for different classes of organisms.

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, ␤-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that ␤-glucuronidase entered the cell ϳ3-4-fold faster than IGF-II. Unlabeled ␤-glucuronidase stimulated the rate of internalization of 125 I-IGF-II to equal that of 125 I-␤-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize ␤-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with ␤-glucuronidase generated a complex composed of two receptors and one ␤-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization.The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) 1 is a type I transmembrane glycoprotein that cycles through the Golgi, endosomes, and the plasma membrane to carry out its role in the biogenesis of lysosomes and in the clearance of the polypeptide insulin-like growth factor II (IGF-II) (1, 2). In the Golgi, the receptor binds newly synthesized acid hydrolases modified with mannose 6-phosphate (Man-6-P) residues on their asparagine-linked oligosaccharides and transports them to endosomes via clathrincoated vesicles (3-5). The acid hydrolases are released in the acidified endosome and then packaged into lysosomes while the receptor either returns to the Golgi to bind another ligand or moves to the plasma membrane (6, 7). At the plasma membrane, the M6P/IGF-II receptor mediates internalization of Man-6-P-containing ligands and IGF-II (3,5,8).The interactions of IGF-II and Man-6-P-containing ligands with the M6P/IGF-II receptor have been characterized in several studies (8 -12). The extracellular portion of the M6P/ IGF-II receptor contains 15 homologous repeating domains of ϳ147 amino acids each (13). Domains 3 and 9 (numbering from the amino terminus) each bind 1 mol of Man-6-P, and the single IGF-II-binding site has been mapped to domain 11 in the extracellular region (14 -16). Man-6-P residues do not inhibit binding of IGF-II to the receptor, verifying that the two ligandbinding sites are distinct. However, proteins containing Man-6...

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) synthesis. PtdIns(4,5)P 2