No abstract

This study assessed the interactive effects of carbohydrate type (fibre vs starch) and fatty acid (FA) supplementation (0% vs 6% calcium soaps of palm oil FA) on the post-fertilisation development of oocytes recovered from low and moderate body condition score (BCS) heifers. A secondary objective was to compare the FA composition of plasma to that of granulosa cells (GCs) and cumulus -oocyte complexes (COCs) from these animals, and to relate these findings to the developmental potential of oocytes. Plasma, GCs and COCs were recovered from 32 heifers on day 5 of a synchronised oestrous cycle for FA analyses. Oocytes were also recovered on days 10 and 15 of the same cycle after short-term ovarian stimulation (FSH 1 GnRH), and matured, fertilised and cultured to the blastocyst stage in vitro. High levels of dietary starch increased (P < 0.01) plasma insulin but, together with dietary FA, reduced (P < 0.05) blastocyst yields in low, but not in moderate, BCS heifers. Dietinduced alterations to the FA content of plasma were less apparent in GCs and COCs. In summary, although dietary lipids increased the FA content of COCs, the selective uptake of saturated FAs at the expense of mainly polyunsaturated FAs within the follicular compartment ensured that the FA composition of COCs was largely unaffected by diet. However, the concentration of saturated FAs within COCs was inherently high, and so further increases in FA content may have impaired postfertilisation development. The data establish a robust nutritional framework for more detailed studies into the mechanistic effects of dietary composition on the post-fertilisation developmental potential of oocytes.

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 ؎ 51 nM. Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKC␣-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals.

1. Preen oil derived from the preen gland has previously been shown to differ in its composition between birds of different ages, sex and diet. As a part of a larger study on preening behaviour and its components, the relative percentages of fatty acids in preen oil were examined in laying hens that differed in age, beak trimmed status, feather pecking status (pecker and pecked) and method of sampling (either direct from the preen gland or from lipid extracted from feathers). 2. Five experiments are described. Fatty acids extracted from lipid were analysed by gas chromatography (GC). Fatty acid composition was affected by age, by whether or not a bird was feather pecked (but not if it was a feather pecker) and by lipid source. 3. Changes in preen oil composition with age (shown here) may be due to changes in circulating concentrations of hormones. Differences in preen oil composition between feather pecked and non-pecked birds may influence plumage odour and therefore taste, making the plumage of some birds more attractive to pecking than others. 4. The proportions of some fatty acids derived from feathers differed to those extracted from the preen gland. Lipid found on feathers is most likely a combination of that from the preen gland and from sebaceous secretions from the skin.

ADP-ribosylation factors (ARFs) play important roles in both constitutive and regulated membrane trafficking to the plasma membrane in other cells. Here we have examined their role in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. These cells express ARF5 and ARF6. ARF5 was identified in the soluble protein and intracellular membranes; in response to insulin some ARF5 was observed to re-locate to the plasma membrane. In contrast, ARF6 was predominantly localized to the plasma membrane and did not redistribute in response to insulin. We employed myristoylated peptides corresponding to the NH 2 termini of ARF5 and ARF6 to investigate the function of these proteins. Myr-ARF6 peptide inhibited insulin-stimulated glucose transport and GLUT4 translocation by ϳ50% in permeabilized adipocytes. In contrast, myr-ARF1 and myr-ARF5 peptides were without effect. Myr-ARF5 peptide also inhibited the insulin stimulated increase in cell surface levels of GLUT1 and transferrin receptors. Myr-ARF6 peptide significantly decreased cell surface levels of these proteins in both basal and insulin-stimulated states, but did not inhibit the fold increase in response to insulin. These data suggest an important role for ARF6 in regulating cell surface levels of GLUT4 in adipocytes, and argue for a role for both ARF5 and ARF6 in the regulation of membrane trafficking to the plasma membrane.Insulin stimulates glucose disposal in peripheral tissues by virtue of the expression of the GLUT4 glucose transporter isoform (1-3). In the absence of insulin, this transporter is intracellularly sequestered within the elements of the endosomal system, the trans Golgi network and a specialized storage compartment (4 -7). Upon insulin stimulation or muscle contraction, GLUT4 is re-distributed from these intracellular locations to the plasma membrane, resulting in a dramatic increase in the rate of glucose entry into these tissues (4 -7). Several studies have suggested that the insulin-stimulated translocation of GLUT4 to the plasma membrane is mechanistically akin to the fusion of small synaptic vesicles with the neuronal plasma membrane (reviewed in Ref.2). This has been supported by the identification of a morphologically similar GLUT4 storage compartment within adipocytes, and by the identification of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) 1 located in GLUT4 vesicles (the v-SNAREs cellubrevin and vesicle-associated membrane protein 2) (8 -11) which bind in a highly specific manner to t-SNAREs located in the adipocyte plasma membrane (Syntaxin 4 and Syndet) (8,10,(12)(13)(14). Vesicle-associated membrane protein 2 has been shown to be the predominant v-SNARE that targets small synaptic vesicles to the pre-synaptic plasma membrane by interacting with the cognate t-SNAREs, syntaxin1, and synaptosome-associated protein of 25 kDa (SNAP-25) found on the target membrane (15). Recent studies implicating vesicle-associated membrane protein 2 in insulinstimulated GLUT4 translocation further strengthen the me...