McGrattan P D, Wylie A R, Bjourson A J
The Queen's University of Belfast, Belfast, UK.
J Endocrinol. 1998 Dec;159(3):381-7. doi: 10.1677/joe.0.1590381.
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.
胰岛素对反刍动物新陈代谢的管理而言，如同对非反刍动物一样不可或缺且十分重要。反刍动物对胰岛素的敏感性和/或反应性降低，这一现象可能更多与胰岛素受体有关，而非激素本身。我们使用简并引物和聚合酶链反应（PCR）筛选了一个绵羊cDNA文库，以检测并测序与人类胰岛素受体基因外显子10、11和12相对应的cDNA部分，其中一个36碱基对（bp）的片段（外显子11）可选择性剪接，产生两种功能特性不同的受体亚型，包括对胰岛素的结合亲和力。绵羊cDNA片段（核苷酸671至770）与人类、恒河猴和大鼠的等效片段分别显示出84%、84%和78%的核苷酸同源性。对三只雄性纯荷兰特克塞尔羔羊（实验1）和五只雄性特克塞尔-灰脸杂交羔羊（实验2）屠宰时采集的选定组织（肝脏、背最长肌、头直肌以及网膜、肾周和皮下脂肪）进行逆转录PCR（RT-PCR），结果显示每个组织（包括脾脏；仅实验2）中有两种mRNA产物，对应分子大小为161和197 bp的cDNA，相差36 bp。序列比对表明，该36 bp片段与人类胰岛素受体基因的选择性剪接外显子11区域同源，且与其他物种的该区域高度保守。在纯种特克塞尔基因型中，外显子11（+）亚型在肝脏中的丰度显著高于肾周脂肪、头直肌和背最长肌骨骼肌（P<0.05），也高于皮下和网膜脂肪（P<0.01）。然而，在该绵羊基因型中，各个肌肉和脂肪库中外显子11（+）亚型的丰度并无差异。在杂交特克塞尔基因型中，外显子11（+）亚型在肝脏中的丰度显著高于肌肉（头直肌，P<0.05；背最长肌，P<0.001）、所有三种脂肪（P<0.001）和脾脏（P<0.001）。在杂交基因型中，外显子11（+）亚型在骨骼肌中的丰度高于所有三种脂肪库（P<0.001），而这三种脂肪库中的亚型丰度相似。这种选择性剪接事件的两种产物表达比例的改变，可能决定组织对胰岛素的敏感性和/或反应性，并为组织间营养分配和营养利用的管理提供一种机制，这对于产肉动物组织生长和胴体特性调控至关重要。
