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

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insu-lin-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 induc-tion 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 b -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

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 b-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 insulininduced 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.
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 insulin receptor substrate (IRS) proteins are intracellular adaptor proteins, and they are the main target proteins for the tyrosine kinase of insulin and IGF-I receptors (2,3). After IRS proteins are tyrosine-phosphorylated by the activated insulin and IGF-I receptors, they interact with the signaling molecules and transmit the signals to downstream machinery, including the phosphatidylinositol 3-kinase (PI3K) pathway and the mTOR complex 1 (mTORC1) pathway.
In mammals, IRS-1 and IRS-2 are major substrates for insulin and IGF-I receptors and are widely expressed (2,3). They both contain a pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and a C-terminal region, including the potential residues of tyrosine for phosphorylation by the activated insulin/IGF-I receptor. The PH and PTB domains mediate the associations with the activated insulin/IGF-I receptor (4,5). Despite these substrates' similar structures, the physiological functions of IRS-2 differ from those of IRS-1, as has been demonstrated using knockout (KO) mouse models.
In mice, IRS-2 deficiency does not affect growth, but it causes type 2 diabetes as a result of hepatic insulin resistance and the loss of b-cell function by 10 weeks of age (6)(7)(8). In contrast, mice lacking IRS-1 are growth-retarded at the embryonic and postnatal periods and display insulin resistance (especially in skeletal muscle and adipose tissue), but they do not develop type 2 diabetes because of the adequate insulin secretion from b-cells (9)(10)(11). These findings indicate that IRS-2 and IRS-1 play distinct roles in the regulation of glucose homeostasis; however, the available data are limited to mice due to the difficulties of gene targeting in other animals.
The discovery of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 system made it possible to produce gene knockout models of many species, including rats (12)(13)(14)(15). In the present study, we generated IRS-2 KO rats by using the CRISPR/Cas9 system to determine the physiological functions of IRS-2 in rats.

Results
The generation of IRS-2 knockout rats using the CRISPR/Cas9 system 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. We then chose two male F0 rats that had either a 4,099-bp deletion (mutation 1) or a 363-bp deletion (mutation 2) to establish the colonies (Fig. 1,  A and B). Because these deletions include the start codon, the translation of short-length IRS-2-like protein could occur by using an alternative ATG located downstream of the annotated start codon.
However, the RT-PCR analysis did not detect either fragment 1 (Irs-2_1) or fragment 2 (Irs-2_2) of IRS-2 mRNA in the brains of the homozygous rats with mutation 1 (Fig. 1C), indicating that IRS-2-like mRNA was not expressed in these rats. In addition, the immunoblot analysis of the total protein and that of the immunoprecipitated IRS-2 protein from the brains and epididymal white adipose tissues (epiWAT) of the homozygous rats with mutation 1 confirmed the absence of IRS-2 protein (Fig. 2, A and B). In the brains of the homozygous rats with mutation 2, both fragment 1 (Irs-2_1) and fragment 2 (Irs-2_2) of IRS-2 mRNA expression were apparent (Fig. 1D), and a low level of short-length IRS-2-like protein (approximately 122 kDa) was expressed; this protein could be the product with a deletion of the PH domain and one-half of a PTB domain predicted from the sequence (Fig. 2, C and D). In the epiWAT of the homozygous rats with mutation 2, the truncated IRS-2 protein was also expressed (Fig. 2C), but it was not clearly detected in the immunoprecipitated IRS-2 protein samples (Fig. 2D). The levels of IRS-1 protein in the brain and epiWAT were comparable between the WT and mutant rats of both lines (Fig. 2, A and C). In light of these results, we decided to use the mutation 1 line as KO rats for further experiments.
The disruption of IRS-2 impaired growth At 1-2 days after birth, the neonate KO rats had 25-30% lower body weights (BWs) compared with the WT (WT) and heterozygous (hetero) rats (WT: 7.1 6 1.0 g; hetero: 6.5 6 0.8 g; KO: 5.0 6 0.5 g). Among the males, the lower BWs of the KO rats persisted until after weaning (Fig. 3A), and at 15 weeks of age the BWs of the KO rats were approximately 65% of those of the WT rats (Table S1). During the experimental period, there was no difference in BW between the WT and heterorats.
At 15 weeks of age, the body length was significantly shorter in the male KO rats compared with the male WT rats (Fig. 3, B and C). The weights of the liver, gastrocnemius muscle, kidney, brain, and epidydimal fat pad were significantly lower in the male KO rats compared with the male WT rats (Table S1), and the ratio of these tissues' weights to the BW (%), except for gastrocnemius muscle, were also significantly lower in the male KO rats than in the male WT rats (Fig. 3D). The serum growth hormone (GH) and IGF-I levels were significantly lower in the male KO rats versus the male WT rats (Fig. 4, A and B). The black bars represent the locations of the fragments 1 and 2 of Irs-2 (Irs-2_1 and Irs-2_2), which was detected by the RT-PCR analysis. C and D, the RT-PCR analysis for Irs-2_1, Irs-2_2, and Actb expression in the brain of 15-week-old WT (1/1) rats and homozygous rats with mutation 1 (m1/m1) (C) or mutation 2 (m2/m2) (D).
Among the females, the KO rats also showed attenuated BW gain (Fig. 3E) and shorter body lengths compared with the WT rats, as in the males (Fig. 3, F and G). The weights of the liver, gastrocnemius muscle, kidney, and brain were significantly lower in the female KO rats compared with the female WT rats (Table S1), and the ratio of these tissues' weights to BW (%), except for gastrocnemius muscle, were significantly lower in the female KO rats than in the female WT rats (Fig. 3H). In contrast to the reduced levels of GH and IGF-I in the KO males, the serum GH level was comparable between the female WT and KO rats (Fig. 4C), and the serum IGF-I level was significantly higher in the female KO rats than in the female WT rats (Fig. 4D). A similar reduction of BW was observed in male and female rats carrying mutation 2 despite the incomplete deletion of IRS-2 in that line (Fig. S1).
The disruption of IRS-2 did not impair glucose tolerance or insulin sensitivity IRS-2-deficient mice have exhibited glucose intolerance and insulin resistance by 10 weeks of age (6,7). We therefore performed a glucose tolerance test (GTT) and a insulin tolerance test (ITT) to evaluate the glucose metabolism and insulin sensitivity in 11-to 13-week-old male and female KO rats. During the GTT, the male WT and KO rats showed peak blood glucose levels at 15 min after the glucose administration, and this level was significantly lower in the male KO rats compared with the male WT rats (Fig. 5A). The incremental area under the glucose curve (AUC) during the GTT was slightly but significantly lower in the male KO rats versus the male WT rats (Fig. 5B), indicating that glucose tolerance was slightly enhanced in the male KO rats compared with the male WT rats. The plasma insulin levels before and after the glucose injection tended to be lower in the male KO rats than in the male WT rats, but these differences did not reach significance (Fig. 5, C and D).
During the ITT, the male KO rats had slightly but significantly higher fasting blood glucose levels; however, their blood glucose levels at 120 min after the insulin injection tented to be lower than that observed in the male WT rats (p = 0.07, Fig.  5E). The area above the curve (AAC) for the changes of blood glucose levels during the ITT was significantly higher in the male KO rats than in the male WT rats (Fig. 5F), indicating that insulin sensitivity was enhanced in the male KO rats. Similar GTT and ITT results were observed in the male WT and KO rats at 27-28 weeks of age (Fig. S2).
Unlike the male KO rats, the female KO rats exhibited glucose and insulin tolerance that was similar to that of the female WT rats at 11-13 weeks of age (Fig. 6, A-F), although there Figure 2. The IRS-2 protein expression in the 15-week-old Irs-2-mutated rats. A-D, the IRS-2 protein level in the brain and epiWAT of WT (1/1) rats and homozygous rats with mutation 1 (m1/m1) (A and B) or mutation 2 (m2/m2) (C and D). A and C, total brain and epiWAT homogenates were analyzed by immunoblotting with antibodies against IRS-1, IRS-2, actin, and b-tubulin. Actin and b-tubulin were used as an internal control. A representative immunoblot of IRS-2 was detected using polyclonal anti-IRS-2 antibody (IRS-2 (p)). B and D, the immunoprecipitated IRS-2 from the brain homogenates was analyzed by immunoblotting with polyclonal (IRS-2 (p)) and monoclonal (IRS-2 (m)) anti-IRS-2 antibodies. The immunoprecipitated IRS-2 from the epiWAT homogenates was analyzed by immunoblotting with anti-IRS-2 (IRS-2 (m)) antibody. was a small difference in the changes in the blood glucose level at 15 min during the GTT (Fig. 6A). 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). In the ITT, the changes in the blood glucose level were similar in the WT and KO females, although the calculated AAC was slightly increased in the female KO rats (Fig. S3, E and F).
The disruption of IRS-2 did not change the insulin-induced tyrosine phosphorylation of insulin receptor (IR) and IRS-1 in the liver and skeletal muscle Because the male KO rats showed a small enhancement of insulin sensitivity, we next investigated the early steps of insulin signaling in the liver and skeletal muscle of both WT and KO males. 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). . BW, body length, and organ weights in IRS-2 KO rats. A, the changes of BW in male WT (n = 12), heterozygous (hetero; n = 13), and IRS-2 knockout (KO; n = 11) rats from 3 to 11 weeks of age. B, a male KO rat and a male WT littermate at 15 weeks of age. C, the body length in 15-week-old male WT (n = 13) and KO (n = 12) rats. The body length was measured as the distance from nose tip to anus. D, the weights of the liver, gastrocnemius muscle, kidney, brain, and epiWAT per BW in 15-week-old male WT (n = 13) and KO (n = 12) rats. E, the changes of BW in 3-to 11-week-old female WT (n = 13), hetero (n = 13), and KO (n = 12) rats. F, a female KO rat and a female WT littermate at 15 weeks of age. G, the body length in 15-week-old female WT (n = 13) and KO (n = 12) rats. H, the weights of the liver, gastrocnemius muscle, kidney, and brain per BW in female WT (n = 13) and KO (n = 12) rats. Values are mean 6 S.D. *, p , 0.05 versus WT rats; #, p , 0.05 versus Heterorats.

Disruption of IRS-2 impairs growth in rats
In the liver, insulin significantly increased the tyrosine phosphorylation of insulin receptor b-subunit (IRb) and IRS-1 in both WT and KO rats, but the absence of IRS-2 did not affect the levels of this insulin-induced phosphorylation (Fig. 8, A and B). Similarly, insulin caused comparable activations of IRS-1-associated PI3K in the livers of male WT and KO rats (Fig. 8, C and D).
Similar to the liver, 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). In contrast, a marginally significant reduction of the insulin-stimulated activity of PI3K associated with IRS-1 was seen in the skeletal muscle of the male KO rats compared with the male WT rats (Fig. 8, G and H).
The disruption of IRS-2 enhanced the insulin-induced activation of the mTORC1 pathway in the liver of KO rats We then examined insulin signaling downstream of PI3K in the liver and skeletal muscle of male WT and KO rats. In the liver, insulin significantly increased phosphorylation of Akt, GSK3b, and S6K (Fig. 9, A-D). The absence of IRS-2 did not affect the insulin-stimulated phosphorylation levels of Akt and GSK3b (Fig. 9, A-C), whereas the insulin-stimulated phosphorylation level of S6K, a substrate of mTORC1, was significantly higher in the KO rats than in the WT rats (Fig. 9, A and D). Because the mTORC1/S6K pathway is shown to be involved in the feedback regulation by insulin through serine (Ser)/threonine (Thr) phosphorylation of IRS-1 (16), phosphorylation of IRS-1 at Ser-307, a site phosphorylated by mTORC1/S6K pathway in response to insulin (17)(18)(19)(20), was examined as well as its tyrosine phosphorylation. Insulin significantly increased the Ser-307 and tyrosine phosphorylation of IRS-1 in both WT and KO rats (Fig. 9, E-G). Despite the higher phosphorylation level of S6K in the liver of KO rats (Fig. 9, A and D), insulin-stimu-lated Ser-307 and tyrosine phosphorylation levels of IRS-1 in the liver were comparable with WT rats (Fig. 9, E-G).
In the skeletal muscle, insulin significantly increased the phosphorylation of Akt, G3K3b, and S6K in the male WT and KO rats (Fig. 10, A-D). Despite the reduced insulin-induced IRS-1-associated PI3K activity, no effect of the deletion of IRS-2 was observed in the levels of these insulin-stimulated phosphorylation (Fig. 10, B-D).

Discussion
In the present study, we created IRS-2 KO rats by using the CRISPR/Cas9 system, and we analyzed the rats phenotypes'. The genetic disruption of 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. Moreover, the male IRS-2 KO rats showed slightly increased insulin sensitivity along with an up-regulation of insulin signaling through the mTORC1 pathway in the liver. These results indicate that in rats, IRS-2 plays critical roles in the regulation of growth but not in the glucoselowering effect of insulin. Similar results were obtained using two independent lines of IRS-2 KO rats (carrying mutation 1 and mutation 2), supporting the concept that these phenotypes were specific for the deficiency of full-length IRS-2 protein.
Both male and female IRS-2 KO rats had lower BWs and shorter body lengths compared with the WT rats during the postnatal period up to 15 weeks of age. Compared with the male WT rats, the male IRS-2 KO rats had lower levels of serum GH and IGF-I, two major hormones regulating growth (21), whereas the female IRS-2 KO rats had similar serum GH levels and rather higher IGF-I levels compared with the female WT rats. These results suggest that the changes in the levels of these hormones were not primary causes of the growth inhibition induced by deletion of the Irs-2 gene in rats. The IGF-I signaling and its actions could be attenuated in IRS-2 KO males and females. In addition, the reduction of the serum levels of GH and IGF-I could have contributed to the more severe growth inhibition phenotype in the males compared with the females.
In addition to the BWs, the weights of the liver, gastrocnemius muscle, kidney, and brain were also lower in both the male and female IRS-2 KO rats compared with the corresponding WT rats. Of these tissues, the weights of the kidney and brain normalized to the BW were much lower in the IRS-2 KO rats than in the WT rats. In contrast, the deletion of IRS-2 had a smaller effect on the normalized liver weights and no effect on the gastrocnemius muscle weights normalized to BW. These findings imply that IRS-2 is important for the control of kidney and brain growth but not for skeletal muscle growth.
Previous studies have shown that IRS-2 KO mice develop type 2 diabetes by 10 weeks of age as a result of hepatic insulin resistance and inadequate compensatory insulin secretion due to impaired b-cell hyperplasia (6,7,(22)(23)(24). From these data, we speculated that the IRS-2 KO rats would also develop type 2 diabetes; however, they did not show glucose intolerance or insulin resistance up to 28 weeks of age. The data showing the changes in the plasma insulin levels during the GTT imply that the insulin secretion in IRS-2 KO rats was nearly normal. These results indicate that the deletion of IRS-2 did not impair glucose tolerance or insulin sensitivity in rats. The islet morphology was also examined, and the results showed that despite the small impacts on the islet size, the total number of islets observed in all sections was fewer in IRS-2 KO rats than in WT rats at 15 weeks of age (Fig. S4) (supporting Materials and Methods). Collectively, these results suggest that IRS-2 KO could maintain normal insulin secretion with fewer pancreatic islets compared with the WT rats.
In addition, in the male rats, deletion of the Irs-2 gene yielded a subtle but significant enhancement of insulin sensitivity together with increased activation of the hepatic mTORC1 pathway in response to insulin. We also examined whether the enhancement of insulin-induced activation of the mTORC1 pathway could increase Ser/Thr phosphorylation of IRS-1 as the feedback regulation of insulin in the liver of IRS-2 KO rats. Because Ser/Thr phosphorylation of IRS-1 is shown to inhibit its tyrosine phosphorylation and mediate insulin resistance (16,25), it is possible that the enhanced mTORC1 pathway attenuates insulin signaling via IRS-1 in the liver of IRS-2 KO rats. Thus far, many Ser/Thr phosphorylation sites of the IRS-1 protein have been reported (16,25). Of these sites, we analyzed phosphorylation of IRS-1 at Ser-307 that is known to be regulated by the mTORC1 pathway in response to insulin (17)(18)(19)(20). The data showed that the absence of IRS-2 did not affect insulin-induced Ser-307 phosphorylation of IRS-1 in the liver as well as its tyrosine phosphorylation, suggesting that enhancement of insulin-induced activation of the mTORC1 pathway is not involved in the negative-feedback regulation of insulin Figure 5. GTT and ITT in 11-to 13-week-old male WT and IRS-2 KO rats. A-D, the GTT results for the 11-to 12-week-old WT (n = 11) and KO (n = 11) rats. A, the changes in blood glucose levels during the GTT. B, the incremental AUC for the blood glucose level during the GTT. C, the changes in plasma insulin levels during the GTT. D, the incremental AUC for the plasma insulin level during the GTT. E and F, the ITT results of the 12-to 13-week-old WT (n = 10) and KO (n = 12) rats. E, the changes in the blood glucose levels during the ITT. F, the AAC for the blood glucose level during the ITT. Values are mean 6 S.D. *, p , 0.05 versus WT rats.

Disruption of IRS-2 impairs growth in rats
signaling via IRS-1. Taken together, the enhanced hepatic mTORC1 pathway in response to insulin could contribute to the increase in insulin sensitivity in IRS-2 KO rats.
The overall phenotypes of the IRS-2 KO rats showed the difference between the sexes. The growth defect induced by the lack of IRS-2 was more severe in males than in the females. The modifications of glucose tolerance between the sexes were also distinct. Despite the normal glucose tolerance in the male and female IRS-2 KO rats, the plasma insulin level during the GTT was lower in the male IRS-2 KO rats than in the male WT rats, but it was higher in the female IRS-2 KO rats than in the female WT rats, especially at older ages. The male IRS-2 KO rats exhibited small but significant enhancement of insulin sensitivity and a higher level of insulin-induced S6K phosphorylation in the liver compared with the male WT rats, whereas the female IRS-2 KO rats did not exhibit these alterations ( Fig. 6 and Fig. S5). Sexual dimorphism has also been observed in IRS-2 KO mice; male IRS-2 KO mice developed type 2 diabetes, whereas female IRS-2 KO mice did not (7). In addition, female IRS-2 KO mice are infertile because of the defects in the hypothalamic reproductive endocrine system, but this phenotype has not been observed in males (26). Further studies are necessary to identify the reasons for the sex differences induced by deletion of the Irs-2 gene in these animal models.
Although the effects of Irs-2 gene deletion on body growth and glucose metabolism differ between rats and mice, smaller than normal brain sizes were commonly observed in both rats and mice lacking IRS-2. In IRS-2 KO mice, all brain regions Figure 6. The GTT and ITT results of the 11-to 13-week-old female WT and IRS-2 KO rats. A-D, the GTT results for the 11-to 12-week-old WT (n = 9) and KO (n = 12) rats. A, the changes in blood glucose levels during the GTT. B, the incremental AUC for the blood glucose level during the GTT. C, the changes in plasma insulin levels during the GTT. D, the incremental AUC for the plasma insulin level during the GTT. E and F, the ITT results for the 12-to 13-week-old WT (n = 11) and KO (n = 12) rats. E, the changes in blood glucose levels during the ITT. F, the AAC for the blood glucose level during the ITT. Values are mean 6 S. D. *, p , 0.05 versus WT rats.
were smaller than those of WT mice, but the brain structure was normal (27). The functions of brain IRS-2 have been elucidated by using tissue-specific IRS-2 KO mice (28). However, the roles of IRS-2 in neurodegeneration and cognition are a matter of controversy. Some studies showed that the lack of brain IRS-2 delayed the progression of neurodegeneration and increased hippocampal memory formation (29,30), whereas other studies indicated that it caused an impairment of Nmethyl-D-aspartate receptor-dependent synaptic plasticity and long-term potentiation in hippocampal neurons (31,32). The IRS-2 KO rats and the future generation of brain-specific IRS-2 KO rats are likely to be useful in studies designed to clarify these contradictions.
In conclusion, our present data demonstrate that in rats, the genetic deletion of IRS-2 reduced body growth and did not impair glucose tolerance or insulin sensitivity. These data indicate that IRS-2 is important in rats for the regulation of growth but is not essential for the glucose-lowering effects of insulin. Further studies using a variety of animal models targeting the IRS proteins are necessary to determine the proteins' physiological functions and their roles in insulin/IGF-I signaling.

Experimental procedures
Animals For all of the animal experiments, the rats were kept in a room maintained at 22 6 2°C with relative humidity at 40-60% and a 12-h light (8:00-20:00) and 12-h darkness (20:00-8:00) cycle. They were allowed free access to water and a standard chow (MF; Oriental Yeast, Tokyo). All experimental procedures were in accord with the guidelines of the Animal Usage Committee of Nippon Medical School and were verified by the Committee.
The generation of IRS-2 mutant rats with the CRISPR/Cas9 system IRS-2 mutant rats were generated with the CRISPR/Cas9 system. The design of single guide (sg) RNA for targeting the rat Irs-2 gene and the preparation of sgRNA and Cas9 mRNA were conducted as described (33). The sequence of sgRNA is shown in Fig.  1A. The injection of Cas9 mRNA and sgRNA was performed as described with some modification (34). Briefly, 25 IU of equine chorionic gonadotropin was intraperitoneally injected into 4-to 5-week-old sexually immature female Wistar-Imamichi rats Figure 7. The insulin-simulated tyrosine phosphorylation of IRS-2 and activation of PI3K associated with IRS-2 in the liver and skeletal muscle of male WT and IRS-2 KO rats. The 8-week-old male WT and KO rats were fasted overnight, and insulin or vehicle was subsequently injected into the inferior vena cava. The liver was dissected at 1 min and skeletal muscle was dissected at 2 min after the insulin injection. A, representative immunoblots of the insulin-stimulated tyrosine phosphorylation of IRS-2 in the liver. The immunoprecipitated IRS-2 from the liver homogenates was analyzed by immunoblotting with anti-phosphotyrosine (pTyr) and monoclonal anti-IRS-2 (IRS-2(m)) antibodies. B and C, the IRS-2-associated PI3K activity in the liver. B, a representative autoradiograph. C, the quantification of radioactivity incorporated into phosphatidylinositol (PIP). D, representative immunoblots of the insulin-stimulated tyrosine phosphorylation of IRS-2 in the skeletal muscle. The immunoprecipitated IRS-2 from the skeletal muscle homogenates was analyzed by immunoblotting with anti-pTyr and anti-IRS-2 (IRS-2(m)) antibodies. E and F, the IRS-2-associated PI3K activity in the skeletal muscle. E, a representative autoradiograph. F, the quantification of radioactivity incorporated into PIP. Values are mean 6 S.D. WT and KO with 2insulin groups, n = 5. WT and KO with 1insulin groups, n = 6. *, p , 0.05 versus WT rats with 2insulin.
Disruption of IRS-2 impairs growth in rats (The Institute for Animal Production, Ibaraki, Japan) to promote follicle maturation. At 48 h after the equine chorionic gonadotropin injection, these rats were intraperitoneally injected with 25 IU of human chorionic gonadotropin to induce ovulation and mated with male Wistar rats (Charles River Japan, Kanagawa, Japan) overnight. The pseudopregnant Wistar-Imamichi rats were prepared by manual vaginocervical stimulation with a glass rod at the proestrous stage. The next day, superovulated zygotes were collected from the female rats' oviducts, treated with hyaluronidase (Sigma-Aldrich), washed, and cultured in M2 medium (Sigma-Aldrich) until the microinjection: approximately 4 pl of a mixture of 50 mg/ml of sgRNA and 50 mg/ml of Cas9 mRNA were injected into the zygotic cytosol with a microinjector (Narishige, Tokyo). After injection, the zygotes were recovered in M16 medium (Sigma-Aldrich) for 1 h and transferred to the oviductal ampullas of the pseudopregnant Wistar-Imamichi rats. Figure 8. The insulin-simulated tyrosine phosphorylation of IRb and IRS-1 and the activation of PI3K associated with IRS-1 in the liver and skeletal muscle of male WT and IRS-2 KO rats. The 8-week-old male rats were treated as described in the legend to Fig. 7. A and B, insulin-stimulated tyrosine phosphorylation of IRb and IRS-1 in the liver. The immunoprecipitated IRb or IRS-1 from the liver homogenates was analyzed by immunoblotting with anti-phosphotyrosine (pTyr), anti-IRb, and anti-IRS-1 antibodies. A, representative immunoblots. B, the ratio of pTyr to the amount of immunoprecipitated IRb (upper graph) or IRS-1 (lower graph). The immunoreactivity of pTyr was quantified and divided by the immunoreactivity of IRb or IRS-1. C and D, insulin-stimulated PI3K activity in the liver. C, a representative autoradiograph. D, the quantification of radioactivity incorporated into PIP. E and F, insulin-stimulated tyrosine phosphorylation of IRb and IRS-1 in skeletal muscle. E, representative immunoblots. F, the ratio of pTyr to the amount of immunoprecipitated IRb (upper graph) or IRS-1 (lower graph). G and H, insulin-stimulated PI3K activity in skeletal muscle. G, a representative autoradiograph. H, the quantification of radioactivity incorporated into PIP. Values are mean 6 S.D. WT and KO with 2insulin groups, n = 5. WT and KO with 1insulin groups, n = 6. The two-way ANOVA results are next to the graph (*, p , 0.05, NS, not significant). Significant differences (p , 0.05) between values are indicated with different letters (a, b, etc.) when there was a significant interaction of genotype and insulin.
After F0 rats were born, PCR was performed with the genomic DNA extracted from the rats' tail tips. The purified PCR products were directly sequenced or sequenced after TA cloning to determine the mutations in Irs-2 gene. We checked the off-target effects in F0 rats by PCR and sequencing analyses and confirmed that the rats did not have any mutations in the potential off-target sites. We selected two male F0 rats that had either mutation 1 or 2 (Fig. 1B) for the expansion of colonies.

Genotyping and breeding
Rats were genotyped by PCR. The primer sequences used were as follows: 59-AGCGGCCACAAGACAGTGTG-39 and 59-TGT-GGCAGCTGTTGCTGGAG-39 for the line of mutation 1; 59-CTCGGTGCGCGATGTGTTAC-39 and 59-GACGGCTGTTC-GCAATTGAG-39 for the line of mutations 1 and 2. All rats used in this study were maintained on a Wistar 3 Wistar Imamichi hybrid background. Male and female heterozygous rats having ei-ther mutation 1 or 2 were intercrossed to obtain homozygous littermates. To perform each experiment using enough numbers of each genotype of rats, approximately 20 mating pairs were set up simultaneously, and several littermates were pooled.
At 15 weeks of age, after having fasted for 4-5 h, WT and homozygous rats having either mutation 1 or 2 were anesthetized with isoflurane (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), and their blood from the carotid artery and tissues were collected. The blood was allowed to clot overnight at 4°C and then subjected to centrifugation at 2,700 3 g for 10 min at 4°C. Serum samples were stored at 280°C until analysis. The excised tissues were frozen immediately in liquid nitrogen and stored at 280°C until use.

The measurement of serum GH and IGF-I levels
The serum GH level was measured by a rat/mouse GH ELISA (ELISA) kit (catalog number EZRMGH-45K, EMD Millipore, Billerica, MA). The serum IGF-I level was measured Disruption of IRS-2 impairs growth in rats by a mouse/rat IGF-I Quantikine ELISA kit (R&D Systems, Minneapolis, MN).

Glucose and insulin tolerance tests
A GTT was performed with 11-to 12-week-old rats. All rats were fasted for 4-5 h and then intraperitoneally injected with glucose solution (1.2 mg/kg BW). Approximately 30 ml of blood was obtained from tail vein using a heparinized microhematocrit capillary tube at 0, 15, 30, 60, and 120 min after glucose injection. The obtained blood sample was subjected to centrifugation at 2,700 3 g for 10 min at 4°C. Plasma samples were stored at 280°C until analysis.
An ITT was performed with 12-to 13-week-old rats. All rats were fasted for 3-4 h and then intraperitoneally injected with bovine insulin (0.5 units/kg BW; Sigma-Aldrich). Blood was obtained from the tail vein at 0, 30, 60, 90, and 120 min after the insulin injection.
The blood glucose levels were measured with a glucometer (Ascensia Breeze, Bayer Medical, Leverkusen, Germany). Plasma insulin levels were measured by an ultra-sensitive mouse/rat insulin ELISA kit (Morinaga, Yokohama, Japan). We used the trapezoidal rule to calculate the incremental AUC for the changes in both blood glucose and plasma insulin levels during the GTT and the AAC for the changes in blood glucose levels during the ITT.

In vivo insulin stimulation
For the in vivo stimulation of insulin signaling, 8-week-old male rats were fasted overnight (14-16 h) and anesthetized with isoflurane (Fujifilm Wako). The abdominal cavity was then opened, and PBS or bovine insulin (0.5 units/kg of BW; Sigma-Aldrich) diluted in PBS was injected into the inferior vena cava. For our analysis of the early steps of insulin signaling, the liver was dissected at 1 min after insulin injection, and the skeletal muscle was dissected at 2 min after insulin injection.
To analyze the downstream of insulin signaling, we prepared a separate set of rats, and the liver and skeletal muscle were dissected at 15 min after insulin injection. The dissected tissues were immediately frozen in liquid nitrogen and stored at 280°C until analysis.

The preparation of protein extracts from tissues
The frozen tissues were homogenized with a Polytron homogenizer in ice-cold homogenizing buffer (50 mM HEPES-NaOH, pH 7.6, 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, 100 KIU/ml of aprotinin, 2 mM EDTA, and 2% Triton X-100). The homogenates were centrifuged at 100,000 3 g for 1 h at 4°C, and the supernatants were collected as protein extracts.

Immunoblotting
Equal amounts of protein sample were subjected to SDS-PAGE, and immunoblotting was performed as described (35). The bands of immunoreactive proteins were detected with an ECL kit (PerkinElmer Life Sciences, Boston, MA) and quantified using a cooled CCD camera system, the LAS-3000 mini (Fujifilm, Kanagawa, Japan). Anti-IRb ( To detect IRS-1 and IRS-2 protein in total protein extracts from the brain and epiWAT, 100 mg of protein sample was subjected to SDS-PAGE. After transblotting, the membranes were blocked with a blocking buffer (5% nonfat milk in TBS with Tween 20 (TBS-T: 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20)) at room temperature for 1 h. The membranes were then incubated with anti-IRS-2 (H-205; Santa Cruz Biochemistry) antibody diluted in Can Get Signal ® Solution 1 (TOYOBO, Osaka, Japan) at 4°C overnight. After washing with TBS-T, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ) diluted in Can Get Signal ® Solution 2 (TOYOBO) at room temperature for 2 h, and washed again with TBS-T. The bands of immunoreactive IRS-1 and IRS-2 were detected as described above.

Statistical analyses
All values are given as the mean 6 S.D. When we compared two groups, the results were analyzed using unpaired Student's t test. The results for the changes of body weight, blood glucose level, and insulin level were analyzed by a two-way repeated measure analysis of variance (ANOVA). The results for insulin signaling were analyzed by a two-way factorial ANOVA. The Tukey-Kramer post-hoc test was performed when a significant interaction between factors was revealed by the two-way ANOVA. Differences were considered significant at p , 0.05.

Data availability
All data are contained within the article.