Effect of plane of nutrition and growth hormone treatment on insulin receptor gene expression and kinase activity of sheep muscle and adipose tissue.

Abstract

In growing animals muscle growth initially takes precedence over adipose tissue accretion [I]. The mechanisms responsible for this are unknown, but tissue specific adaptation of the insulin signalling system could be involved. We have investigated this possibility by varying growth rate in young sheep by feedmg different levels of nutrition and treating chrol4cay. with growth h o n e (GH) and determining effects on insulin receptor kinase activity and receptor protein and mRNA concentration. Thirty-two castrated male lambs of approximately 6 months of age and weighmg between 30 and 40kg were randomly assigned to four treatment groups each of which was fed varying amounts of a lamb fattener diet (55% barley, 35% oats, 2.5% soya; metabolisable energy 12.4 MJ/kg DM and a crude protein content of 112gkg DM) to achieve growth rates of either 50, 100,200 or 350glday over a 3 week period. During the last week, half of the lambs of each group were injected daily for 7 days with GH (O.2mgkgI Liuey, Somidobove, lot 505ALO). Lambs were killed by electrostatic stunning followed by exsanguination. Samples of skeletal muscle (Vastus lateralis) and perirenal adipose tissue were taken and . d a t e l y frozen in liquid N2 prior to membrane preparation and RNA isolation. Muscle membranes were prepared by homogenisation of powdered tissue in 5OmM Tris-HCI, pH 7.4 containing 5mM EDTA, 2pg/mI leupeptin, 5pdmI soyabean trypsin inhibitor, 15pdmI h a m i d i n e , using a polytron. An equal volume of 1M KCI was added, s the sheep insulin -tor CD was 92.6% and 86.0% similar to the comsponding regions of the human [8] and rat [9] insulin receptor cDNAs. Mcasurancnt of insulin receptor mRNA was determined by an RNase protection assay using standard protocols [ 101. Briefly, an antiscnsc transaipt was generated from pSIR restricted with EcoRl using SP6 RNA polymerase and then hybridiscd with 40pg samples of RNA. The produds ofthe hyhdmhon wcrc digested with RNase A and RNase T, and then precipitated with 5% (w/v) hichloroacetic acid onto GFK filters and their radioactivity determined by liquid scintillation counting.