Transgenic mice overexpressing human G972R IRS-1 show impaired insulin action and insulin secretion.

Molecular scanning of human insulin receptor substrate (Irs) genes revealed a single lrs1 prevalent variant, a glycine to arginine change at codon 972 (G972R); previous in vitro studies had demonstrated that the presence of this variant results in an impaired activation of the insulin signalling pathway, while human studies gave controversial results regarding its role in the pathogenesis of insulin resistance and related diseases. To address in vivo impact of this IRS-1 variant on whole body glucose homeostasis and insulin signalling, we have generated transgenic mice overexpressing it (Tg972) and evaluated insulin action in the liver, skeletal muscle and adipose tissue and assessed glucose homeostasis both under a normal diet and a high-fat diet. We found that Tg972 mice developed age-related glucose and insulin intolerance and hyperglycaemia, with insulin levels comparatively low. Glucose utilization and insulin signalling were impaired in all key insulin target tissues in Tg972 mice. There were no differences in pancreatic morphology between Tg972 and wild-type mice, however when insulin secretion was evaluated in isolated islets, it was significantly reduced in Tg972 mice islets at any glucose concentration tested. Under a high-fat diet, Tg972 mice had increased body and adipose tissue weight, and were more prone to develop diet-induced glucose and insulin intolerance. So, we believe that Tg972 mice may represent a useful model to elucidate the interaction between genetic and environmental factors in insulin resistance pathogenesis. Furthermore, they may become an important tool to test novel tailored therapies.

autophosphorylation (10). On the whole, the data obtained in vitro and in cellular models confirm that the presence of the G972R variant is detrimental for insulin signalling, thus suggesting that it may contribute to insulin resistance in human carriers. Data from human studies, however, are more controversial, with some earlier reports describing an association of the G972R variant with type 2 diabetes [3,4,11] and subsequent larger studies not confirming these findings [12][13][14][15]. Interestingly one of this studies [15] has shown that while the presence of the R allele had no independent effect on type 2 diabetes risk, it increased the risk in subjects with elevated fasting glucose and body mass index, thus hinting to the possibility that the G972R variant may have an additive effect with other, genetic or environmental, risk factors in determining the susceptibility to type 2 diabetes and related diseases. To address in vivo the impact of the G972R IRS-1 variant on whole body glucose homeostasis and insulin signalling, we have generated transgenic mice with a generalized overexpression of this IRS-1 form.

Animal husbandry, genotyping and analytical procedures
The human G972R IRS-1 variant was subcloned into a pCAGGS vector and Tg972 mice were generated on an FVB background according to standard procedures [16]. Founders were identified by Southern Blot of tail DNA and the presence of the G972R variant was confirmed by PCR amplification of the same DNA samples and subsequent digestion with MvaI restriction enzyme (Roche Applied Science, Basel, Switzerland) as described [4]. Routine genotyping was then carried out by PCR using specific primers for the human IRS-1 transgene (forward 5´-CTTCTGTCAGGTGTCCATCC-3á nd reverse 5´-TGGCGAGGTGTCCACGTAGC-3´). High Fat Diet (60%Kcal from fat, 20%Kcal from carbohydrates, 20%Kcal from proteins) and normal chow were purchased from Research Diets (NY,USA). Weight gain was measured weekly. Three-to-six months-old male mice were used for the experiments described. Animal studies were approved by the University of Rome 'Tor Vergata' Animal Care and Use Committee and were in accordance to the Principles of Laboratory Animals Care (NIH publication no. 85-23, revised 1985) and to Italian law 116 of January 27, 1992 and following amendments.

In vivo insulin stimulation
After 6-hrs fasting, WT and Tg972 mice were anaesthetized by pentobarbital intraperitoneal injection (40 mg per Kg body weight). Insulin stimulation was carried out as described [17], briefly 5U of regular human insulin were injected into the inferior vena cava, 3 min. later tissues were harvested and frozen in liquid nitrogen.

Western blots
Lysates from white adipose tissue, liver and muscle were prepared and western blots performed as previously described [17]. For immunoprecipitation, tissue lysates were incubated for 16 hrs at 4°C with anti-IRS-1 or anti-IRS-2 antibody. Immunocomplexes were collected with incubation with Protein A-Sepharose for 2 hrs at 4°C. Immunoprecipitated proteins or tissue lysates were then re-suspended in Laemmli buffer, resolved on SDS-PAGE, transferred onto nitrocellulose membranes, and probed with the appropriate antibodies.

2-DOG incorporation into liver glycogen and liver glycogen measurement during a glucose tolerance test
Frozen liver (50-75 mg) was pulverized with mortar and pestle in liquid nitrogen and digested. Macromolecules were then allowed to precipitate and used to determine radioactivity incorporation as described previously.

To determine 2-Deoxy-Glucose (2-DOG) incorporation into liver glycogen, [ 3 H]radioactivity in glycogen counts (degenerations per minute) was divided by the integrated glucose-specific activity area under the curve and
corrected for sample protein content [17].

Pancreata histological analysis and morphometry
Pancreata were fixed overnight in 4% paraformaldehyde and embedded in paraffin using standard techniques [19]. Sections were then placed on glass slides, deparaffinized with xylene and stained with haematoxylin and eosin. Immunostaining for insulin or glucagon was carried out with the Histomouse-SP kit (Zymed Laboratories Inc., South San Francisco, California, USA) according to the manufacturers' instructions. Six sections for each pancreas were imaged at a 20x magnification and quantified for surface areas. Areas corresponding to the ␤-cells were measured by dividing the area of insulin-positive immunoreactivity by whole pancreas area.

Pancreatic insulin content
To assess insulin content, pancreata were extracted in 400 µl of acid ethanol extraction medium overnight at 4°C [20]. All extracts were stored at -20°C for subsequent insulin levels detection.

Islet isolation and insulin secretion
Islets were isolated using the intraductal collagenase technique as described [21]. Briefly, the common bile duct was cannulated in the anterograde direction and the pancreas distended with collagenase type P (4 mg in 5

Results
To investigate in vivo the impact of the human G972R IRS-1 variant on insulin resistance and glucose homeostasis, a cDNA containing this mutant form was subcloned into a pCAGGS vector and used to generate transgenic mice (Tg972). IRS-1 protein overexpression was confirmed by Western Blot in protein lysates from liver (Fig. 1A), skeletal muscle (Fig. 1B), adipose tissue (Fig. 1C), heart (Fig. 1D), pancreas (Fig. 1E) and purified pancreatic islets (Fig. 1F)  Reduced insulin secretion and altered insulin sensitivity may both play a role in the observed phenotype. To explore the first possibility, we evaluated islets morphology and insulin content in 4-to 6-months-old male mice. We did not observe any difference either in islets number and size or in the structure of the islets, as assessed by insulin and glucagon staining, ( Fig. 2A-E) or in the total pancreatic insulin content (3.445±2.41 and 3.875±2.49 ng insulin/microgram protein for WT and Tg972 mice, respectively) of Tg972 mice compared with their WT littermates. However, when we assessed insulin secretion in isolated islets, we observed that insulin release was significantly reduced in Tg972 islets at any glucose concentration tested (Fig. 2F,  P<0.0001). Then, we proceeded to evaluate peripheral insulin resistance and observed a 42% (P<0.01) and a 40% (P<0.05) reduction in glucose uptake in skeletal muscle and adipose tissue of Tg972 mice, respectively, as compared with WT littermates (Fig. 3A and B) and a 42% reduction in glucose incorporation into glycogen, an index of glycogen synthesis, (Fig. 3C, P<0.0001) in the liver of Tg972 mice.
These data suggest that the phenotype of 6-months-old Tg972 mice was indeed due to reduced insulin secretion, paralleled by increased demand from insulin resistant peripheral tissues. To further elucidate the molecular mechanisms underlying this phenotype, we assessed the activation of key steps in insulin signalling in peripheral target tissues from 3-4-months-old mice. We observed that, despite the increased IRS-1 expression and total IRS-1 tyrosine phosphorylation (Fig. 4A and B), the amount of PI3-kinase regulatory subunit, p85, co-precipitated with IRS-1 was significantly reduced in liver lysates from Tg972 mice ( Fig. 4A and C, P<0.0001 (Fig. 4F). The reduced activation of the upstream insulin signalling steps observed in Tg972 mice was paralleled by a decreased phosphorylation of Akt, a key transducer of downstream insulin signalling in the liver (Fig. 5A, P<0.01), skeletal muscle (Fig. 5B,  P<0.01) and adipose tissue (Fig. 5C, P<0.05). Furthermore in the liver, we observed also a 56% reduction of the phosphorylation of glycogen synthase kinase-3 (GSK-3), an Akt substrate, which plays a pivotal role in the regulation of glycogen synthesis (Fig. 5D, P<0.05).  Finally, to study the effects of environmental factors, we characterized the metabolic phenotype of Tg972 under a high-fat diet (HFD) as a model of environmental challenge [22]. Under HFD, weight gain was significantly increased in Tg972 mice compared with their WT littermates (Fig. 6A, P<0.0001). Consistently, epidydimal adipose tissue weight was significantly increased in HFD Tg972 mice compared with WT littermates (P<0.05, Fig. 6B). The increased adipose tissue weight was mirrored by altered expression of genes involved in adipose tissue function such as leptin, resistin, peroxisome proliferator activated receptor-␥ (PPAR␥) and adiponectin (P<0.05, Fig. 6C-F). Interestingly, HFD accelerated the insurgence of hyperglycaemia (Table 1) and glucose intolerance in Tg972 mice; indeed transgenic mice show a significantly reduced glucose tolerance (P<0.0001, Fig. 6G) and insulin sensitivity (P<0.0001, Fig. 6H) at 3 months of age as compared with their WT littermates. At variance with the phenotype observed under a normal diet, Tg972 mice did not show reduced insulin levels compared with their WT littermates ( Table 1), suggesting that under HFD peripheral insulin resistance may be the prevalent defect responsible of the observed metabolic alterations. However, when HFD was prolonged until the mice were 6-months-old, Tg972 mice showed significantly lower fed and fasting insulin levels (Table 1) paralleled by significantly increased glucose levels ( Table 1), suggesting that, at this stage, they have developed a deficiency in insulin secretion. This hypothesis was sustained by the observation that serum insulin levels 120 min. after glucose injection were significantly lower in Tg972 mice than in their WT littermates (P<0.0001, Fig. 6L), and the Tg972 mice were therefore unable to counteract the rise in glucose levels after a glucose load (P<0.0001, Fig. 6I).

Discussion
To assess the impact of the G972R variant on whole-body glucose homeostasis and insulin signalling, we have generated transgenic mice with a generalized overexpression of this IRS-1 form. We observed that key insulin target tissues from Tg972 mice, such as skeletal muscle, liver and adipose tissues, showed reduced activation of the insulin signalling pathway downstream of IRS-1, similarly to what has been previously showed in primary and immortalized cell lines. Indeed, it has been known for several years that in a number of different cellular systems the presence of the R residue at position 972 in the IRS-1 molecules causes a reduced binding of IRS-1 with PI3-kinase, however until recently the molecular mechanism underlying this phenomenon was not elucidated. Kahn and coworkers demonstrated in in vitro experiments with IRS-1 fragments that the R residue caused a reduction of insulin-stimulated phosphorylation of two flanking tyrosine phosphorylation sites, Tyr 941 and Tyr 989 , that play a pivotal role in IRS-1 binding to PI3-kinase [10]. Here, we were able to confirm for the first time in vivo that in the presence of G972R IRS-1 insulin-stimulated phosphorylation of Tyr 941 and Tyr 989 is significantly reduced. The impaired activation of the insulin pathway resulted in decreased tissue specific insulin action and eventually, as the mice aged, in whole-body insulin resistance. The decreased insulin sensitivity in aged mice was not paralleled by a compensatory increase in plasma insulin levels, thus suggesting that the G972R variant made pancreatic beta-cells unable to respond to the increased insulin demand. Indeed, 6-months-old Tg972 mice showed significantly reduced fed insulin levels and a significantly reduced G/I ratio in a fasting state. Furthermore, islets isolated from Tg972 mice, before the insurgence of insulin resistance, show a reduced glucose-stimulated insulin secretion, suggesting that the two defects, decreased insulin sensitivity and decreased insulin secretion, develop in parallel in the Tg972 mice and contribute to the progression of the disease. Interestingly, HFD accelerates the insurgence of glucose intolerance and insulin resistance in Tg972 mice, thus sustaining the hypothesis that the G972R variant may act as a modifier gene, increasing the risk for type 2 diabetes in individuals that present an unfavourable phenotype [15]. Indeed, the observation that obese carriers of the G972R IRS-1 variant  are characterized by decreased insulin sensitivity associated with a cluster of metabolic alterations, including elevated fasting levels of plasma glucose, had led to hypothesize that this variant amplifies obesity-linked insulin resistance [15,23,24]. The molecular mechanism underlying this interaction however has not been explained yet, therefore the Tg972 mice can represent a useful tool to address this issue. Of note, adipose tissue from Tg972 obese mice express higher mRNA levels of leptin and resistin; interestingly results from microarray studies on adipose tissue obtained from human obese subjects showed that G972R carriers have increased expression of obesity-related genes, including leptin and resistin, as compared with weight-matched WT individuals [25]. This observation suggests that the G972R IRS-1 variant may affect insulin sensitivity through multiple mechanisms, including regulation of gene expression, that have not been exhaustively addressed. In conclusion, we believe that the humanized mouse model we have generated in this study is an ideal model to further elucidate the interaction between genetic and environmental factors in the pathogenesis of type 2 diabetes. Additionally, we have previously shown that type 2 diabetic carriers of the G972R variant had an increased risk for secondary failure to sulfonylureas [26]; thus providing a first evidence that this variant may influence treatment response in carriers. More recently, Haring and coworkers found cerebrocortical insulin resistance in individuals carrying the G972R IRS-1 variant [27]. Therefore, in light of the recent developments in the field of pharmacogenetics [28], the Tg972 mice may become a useful tool to test tailored therapies.