Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats

Yuka Toyoshima,Katsuyuki Nakamura,Reiko Tokita,Naomi Teramoto,Hidetoshi Sugihara,Hisanori Kato,Keitaro Yamanouchi,Shiro Minami

doi:10.1074/jbc.ra120.013095

Yuka Toyoshima, Katsuyuki Nakamura + Show 6 more

Open Access

https://doi.org/10.1074/jbc.ra120.013095

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Abstract

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired β-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.

Highlights

The pleiotropic effects of insulin and insulin-like growth factor (IGF)-I on the regulation of somatic growth and a variety of metabolic functions are mediated by a complex network of intracellular signaling pathways [1]
The genetic disruption of insulin receptor substrate-2 (IRS-2) resulted in the inhibition of growth in rats, but it did not impair glucose tolerance or insulin sensitivity until 28 weeks of age
The male insulin receptor substrate (IRS)-2 KO rats showed slightly increased insulin sensitivity along with an up-regulation of insulin signaling through the mTOR complex 1 (mTORC1) pathway in the liver

Summary

Results

The PCR amplification of targeting loci and the subsequent sequencing analysis confirmed the mutations of the F0 rats, but IRS-2 protein was expressed in all F0 rats because they had an allele of WT, 1-base pair (bp) insertion, or a 3-bp deletion upstream of the start codon of Irs-2 gene. The 27- to 28-week–old female KO rats had significantly higher plasma insulin levels at 15 min after the glucose injection compared with the female WT rats, and they maintained normal blood glucose levels during the GTT (Fig. S3, A–D). Our results first showed that there was no increase in the tyrosine phosphorylation of IRS-2 or in the activation of IRS-2–associated PI3K in the liver and skeletal muscle of KO males (Fig. 7). In skeletal muscle, insulin significantly increased the tyrosine phosphorylation of IRb and IRS-1, and there was no effect of the deletion of Irs-2 gene on the levels of this insulin-induced phosphorylation observed in the male WT and KO rats (Fig. 8, E and F). Despite the higher phosphorylation level of S6K in the liver of KO rats (Fig. 9, A and D), insulin-stimu-

Discussion

Experimental procedures

Genotyping and breeding

Glucose and insulin tolerance tests

In vivo insulin stimulation

The preparation of protein extracts from tissues