A partial cDNA sequence of the ovine insulin receptor gene: evidence for alternative splicing of an exon 11 region and for tissue-specific regulation of receptor isoform expression in sheep muscle, adipose tissue and liver.
Insulin is as integral and important to the management of metabolism in ruminants as it is in non-ruminants. The suggestion of a lowered ruminant sensitivity and/or responsivity to insulin may relate more to the insulin receptor than to the hormone itself. We screened an ovine cDNA library using degenerate primers and polymerase chain reaction (PCR) to detect and sequence a cDNA portion corresponding to exons 10, 11 and 12 of the human insulin receptor gene in which a 36 base pair (bp) segment (exon 11) is alternatively spliced to produce two distinct receptor isoforms differing in functional characteristics including binding affinity for insulin. The ovine cDNA segment (nucleotides 671 to 770) displayed 84, 84, and 78% nucleotide homology to equivalent segments from the human, rhesus monkey and rat respectively. Reverse transcription PCR (RT-PCR) of selected tissues (liver, m. longissimus dorsi, m. rectus capitis and omental, perirenal and subcutaneous fats) taken at slaughter from three male, pure Dutch Texel lambs (experiment 1) and five male Texel-Greyface crossbred lambs (experiment 2) revealed two mRNA products in each tissue (including spleen; experiment 2 only) corresponding to cDNAs of molecular sizes 161 and 197 bp -- a difference of 36 bp. Sequence alignment showed the 36 bp segment to be homologous to the alternatively spliced exon 11 region of the human insulin receptor gene and to be highly conserved with that from other species. The abundance of the exon 11(+) isoform in the purebred Texel genotype was significantly higher in liver than in perirenal fat and rectus capitis and longissimus dorsi skeletal muscles (P<0.05) and higher also than in subcutaneous and omental fats (P<0.01). There was, however, no difference in the abundance of the exon 11(+) isoform between the individual muscle and fat depots in this sheep genotype. The abundance of the exon 11(+) isoform in the crossbred Texel genotype was significantly higher in liver (P<0. 05) than in the muscles (rectus capitis, P<0.05; longissimus dorsi, P<0.001), all three fats (P<0.001) and spleen (P<0.001). In the crossbred genotype, the abundance of the exon 11(+) isoform was higher in skeletal muscle than in all three fat depots (P<0.001), in which the isoform abundance was similar. Altered ratios of expression of the two products of this alternative splicing event could determine tissue sensitivity and/or responsivity to insulin and provide a mechanism for the management of nutrient partitioning and nutrient utilisation between tissues which is fundamental to the growth of tissues and manipulation of carcass characteristics in meat-producing animals.
McGrattan
P D
PD
The Queen's University of Belfast, Belfast, UK.
Wylie
A R
AR
Bjourson
A J
AJ
D000273:Adipose Tissue / Q000378:metabolism; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis; D005091:Exons; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008253:Macaca mulatta; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D020033:Protein Isoforms; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep / Q000235:genetics*
D018076:DNA, Complementary; D020033:Protein Isoforms; D011972:Receptor, Insulin