Pdx1 Expression in Irs2-deficient Mouse β-Cells Is Regulated in a Strain-dependent Manner*

We previously demonstrated that Irs2-/- mice develop diabetes due to β-cell growth failure and insulin resistance; however, glucose-induced insulin secretion was increased in islets isolated from Irs2-/- mice. Pdx-1, a transcription factor important for maintenance of the β-cell function, was recently reported to be severely reduced in Irs2-/- murine β-cells. We report herein that Pdx-1 expression, including the amount of Pdx-1 localized in the nucleus, is not down-regulated in our Irs2-/- murine β-cells with a C57BL/6 background. We have also demonstrated the expression of upstream genes of Pdx-1, such as HNF3β and HNF1α, as well as its downstream genes, including insulin, Glut2, and Nkx6.1, to be well preserved. We have further demonstrated Pdx-1 expression to also be preserved in β-cells of 30-week-old diabetic Irs2-/- mice. In addition, surprisingly, even in Irs2-/- mice on a high fat diet with markedly elevated blood glucose, exceeding 400 mg/dl, Pdx-1 expression was not reduced. Furthermore, we found Pdx-1 to be markedly decreased in certain severely diabetic Irs2-/- mice with a mixed C57BL/6J × 129Sv background. We conclude that 1) Pdx-1 expression in Irs2-/- mice is regulated in a strain-dependent manner, 2) Irs2-/- mice develop diabetes associated with β-cell growth failure even when Pdx1 expression is preserved, and 3) Pdx-1 expression is preserved in severely hyperglycemic Irs2-/- mice with a C57BL/6 background on a high fat diet.

Insulin receptor substrate (IRS) 1 -1 and IRS-2 are two major substrates for insulin receptor tyrosine kinase and insulin-like growth factor (IGF) receptor tyrosine kinase (1)(2)(3)(4)(5)(6). Irs1 Ϫ/Ϫ mice have insulin-resistant skeletal muscles but do not develop diabetes because ␤-cells are able to undergo hyperplasia and secrete more insulin to compensate for the insulin resistance (7,8). In contrast, Irs2 Ϫ/Ϫ mice develop diabetes because ␤-cells failed to undergo hyperplasia in the face of hepatic insulin resistance (9 -12). Whereas ␤-cell mass in Irs2 Ϫ/Ϫ mice was reduced to 83% of that in the wild-type mice at the age of 6 weeks, ␤-cell mass in Irs2 Ϫ/Ϫ mice was significantly reduced to 51% at 12 weeks of age (11). Insulin/insulin-like growth factor signaling through IRS and phosphoinositide 3-kinase has been thought to regulate several aspects of ␤-cell function (13)(14)(15)(16)(17). Besides the impaired ␤-cell proliferation in Irs2 Ϫ/Ϫ mice, ablation of p70 s6k1 , an Akt substrate, is associated with a decrease in ␤-cell size (18). Conversely, overexpression of a constitutively active mutant Akt1 increases ␤-cell mass and protects from streptozotocin-induced diabetes (19,20). However, neither the ablation of p70 s6k1 nor ␤-cell-specific overexpression of Akt1 affects glucose sensing or insulin production (18,20). In fact, we also previously demonstrated glucose-induced insulin secretion to be rather increased in Irs2 Ϫ/Ϫ murine islets (11). In addition, insulin contents of Irs2 Ϫ/Ϫ murine islets were comparable with those of islets from wild-type mice (11), suggesting the ␤-cell functions of insulin synthesis and secretion to be well preserved in Irs2 Ϫ/Ϫ murine islets.
Pdx-1 is a key transcription factor involved in pancreatic development in early embryonic stages (21,22) and is also involved in the maintenance of ␤-cell functions in later embryonic stages (23)(24)(25). Pdx-1 is also reportedly involved in insulin secretory function in both adult mice and cultured ␤-cell lines (25,26). Pdx-1 expression reportedly lies downstream from HNF3␤ and HNF1␣ (27) and regulates insulin, Glut2, Nkx6.1, and glucokinase genes (28 -33). In adult islets, Pdx-1 is required for maintenance of the ␤-cell phenotype, including expression of insulin and Glut2 (34). Pdx-1 is also known as a MODY (maturity onset diabetes of the young) 4 gene (35). Haploinsufficiency of the Pdx-1 gene in mice reportedly results in defects in glucose-stimulated insulin secretion (23,25,26). The Pdx-1 mutation identified in human MODY4 patients may serve as a dominant-negative isoform (36).
Pdx-1 was recently reported to be severely reduced in Irs2 Ϫ/Ϫ murine ␤-cells, suggesting the possibility that IRS-2 directly regulates the expression and function of Pdx-1, thereby maintaining ␤-cell growth and function (37). These studies indicated that Pdx-1 mRNA levels in Irs2 Ϫ/Ϫ islets are reduced to about 20% of those in wild-type islets, associated with HNF3␤ mRNA reduction, and that the protein levels are reduced identically.
However, because glucose-induced insulin secretion was increased and the insulin content was well preserved in our Irs2 Ϫ/Ϫ murine islets (11), we suspected that Pdx-1 is not FIG. 1. Expression of Pdx-1 in pancreatic islets. a, TaqMan RT-PCR of Pdx-1 in islets from 8-week-old Irs2 ϩ/ϩ (n ϭ 6) and Irs2 Ϫ/Ϫ (n ϭ 6) male mice. Total RNA was extracted from each mouse. Data are normalized to the expression of the ␤-actin gene (1), GAPDH (2), and cyclophillin (3). Pdx-1 mRNA levels in Irs2 Ϫ/Ϫ mouse islets were unchanged when ␤-actin and GAPDH were used as internal standard and were increased when cyclophillin was used. N.S., difference not significant. b, Western blots of Pdx-1 in islets isolated from 8-week-old Irs2 ϩ/ϩ and Irs2 Ϫ/Ϫ male mice. For each group, islets from three mice were pooled, and lysates (20 g of protein) were subjected to Western blot analysis with antibodies against Pdx-1 and actin as an internal control. Pdx-1 protein was not decreased in Irs2 Ϫ/Ϫ mouse islets. down-regulated in our Irs2 Ϫ/Ϫ mice. We report herein that Pdx-1 expression, including the amount of Pdx-1 localized in the nucleus, is not down-regulated in our Irs2 Ϫ/Ϫ murine ␤-cells with a C57BL/6J background. In addition, surprisingly, even in Irs2 Ϫ/Ϫ mice on a high fat diet with markedly elevated blood glucose, exceeding 400 mg/dl, Pdx-1 expression was not reduced. Furthermore, we found Pdx-1 to be markedly decreased in some severely diabetic Irs2 Ϫ/Ϫ mice with a mixed C57BL/6J ϫ 129Sv background. We conclude that Pdx-1 expression in Irs2 Ϫ/Ϫ mice is regulated in a strain-dependent manner, and our results suggest that a certain 129Sv-related genetic component(s) is involved in reduced Pdx-1 expression, leading to severe diabetes in Irs2 Ϫ/Ϫ mice.

EXPERIMENTAL PROCEDURES
Animals-IRS-2-deficient mice were generated as previously described (11). Irs2 ϩ/ϩ mice and Irs2 Ϫ/Ϫ mice had a C57BL/6J background, except when specifically stated to have a mixed C57BL/6J ϫ 129Sv background. Mice were housed under a 12-h light/dark cycle and given ad libitum access to food. All experiments in this study were performed on male mice.
Isolation of Islets-Isolation of islets from Irs2 ϩ/ϩ mice and Irs2 Ϫ/Ϫ mice was carried out as described elsewhere (38). In brief, after clamping the common bile duct at a point close to the duodenal outlet, 2.5 ml of Krebs-Ringer bicarbonate buffer (129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 , 5 mM NaHCO 3 , and 10 mM HEPES at pH 7.4) containing 4 mg/ml collagenase (Sigma) was injected into the duct. The swollen pancreas was removed and incubated at 37°C for 3.5 min. The pancreas was dispersed by pipetting and washed twice with ice-cold Krebs-Ringer bicarbonate buffer. RNase inhibitor (Invitrogen) was added in buffer if RNA extraction was required. Islets were manually collected through a stereoscopic microscope.
Immunohistochemistry-The removed pancreata were immediately immersion-fixed in Bouin's solution at 4°C overnight. Tissues were routinely processed for paraffin embedding, and 2-m sections were cut and mounted on silanized slides. With the indicated antibodies, the sections were immunologically stained brown using an Envision Labeled Polymer Kit (DakoCytomation Co. Ltd., Kyoto, Japan) with a DAB Substrate Kit (3,3Ј-diaminobenzidine) (Vector Laboratories, Burlingame, CA) and/or stained red using an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody with Vector Red (Vector Laboratories).
Western Blot Analysis-Isolated islets were homogenized in ice-cold buffer A (25 mM Tris-HCl (pH 7.4), 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride). The protein concen-trations of lysates were equalized for loading by total protein assay (Pierce). SDS samples were separated on polyacrylamide gels and transferred to a Protran nitrocellulose membrane (Schleicher & Schuell). The membrane was incubated with the indicated antisera and horseradish peroxidase-conjugated anti-IgG antibodies. Bands were detected by ECL detection reagents (Amersham Biosciences).
High Fat Diet Experiment-Six-week-old male Irs2 ϩ/ϩ mice and Irs2 Ϫ/Ϫ mice were housed in individual cages. They were divided into two groups, a regular chow group and a high fat diet group. The high fat diet was prepared with the following composition: 32% safflower oil, 33.1% casein, 17.6% sucrose, 1.4% vitamins, 9.8% minerals, 5.6% cellulose, 0.5% DL-methionine, as previously described (40). Materials were purchased from Oriental Yeast Co., Ltd. (Osaka, Japan). Body weight was assessed between 9:00 and 11:00. After 5 weeks of feeding, blood samples were taken from the orbital sinus, and glucose was measured using an automatic blood glucose meter (Glutest Pro; Sanwa Chemical, Nagoya, Japan) between 9:00 and 11:00 in the fed state and after fasting for 16 h from 19:00 to obtain fasted state values. Whole blood was collected and centrifuged in heparinized tubes, and the plasma was stored at Ϫ20°C. Insulin levels were determined using an insulin radioimmunoassay kit (BIOTRAK; Amersham Biosciences) with rat insulin as a standard (40).
Values are presented as means Ϯ S.E. The statistical significance of differences between groups was determined by Student's t test (two-tailed).

RESULTS
Expression of Pdx-1 in Irs2 Ϫ/Ϫ Murine ␤-Cells-Pdx-1 mRNA levels were measured in islets from Irs2 Ϫ/Ϫ mice by real time quantitative PCR. This study did not yield conclusive results, because Pdx-1 mRNA levels in Irs2 Ϫ/Ϫ murine islets were unchanged when ␤-actin and GAPDH were used as an FIG. 3. Expression of genes lying upstream from Pdx-1 in pancreatic islets. a, TaqMan RT-PCR of HNF3␤, HNF1␣, and HNF4␣ in islets from 8-week-old Irs2 ϩ/ϩ (n ϭ 6) and Irs2 Ϫ/Ϫ (n ϭ 6) male mice. Total RNA was extracted from each mouse. Data are normalized to ␤-actin expression. HNF3␤, HNF1␣, and HNF4␣ mRNA expressions were not decreased in Irs2 Ϫ/Ϫ murine islets. N.S., difference not significant. b, Western blots of HNF3␤ in islets from 8-week-old Irs2 ϩ/ϩ and Irs2 Ϫ/Ϫ male mice. For each group, islets from three mice were pooled, and lysates (20 g of protein) were subjected to Western blot analysis with antibodies against HNF3␤ and actin as an internal control. HNF3␤ protein expression was not reduced in Irs2 Ϫ/Ϫ murine islets. internal standard and were increased when cyclophillin was used (Fig. 1a).
Western blot analysis also showed that Pdx-1 protein was not decreased in isolated islets from Irs2 Ϫ/Ϫ mice (Fig. 1b). In addition, immunohistochemical studies revealed Pdx-1 protein to be equally expressed in the nuclei of Irs2 Ϫ/Ϫ murine ␤-cells at 8 weeks of age, as compared with those of Irs2 ϩ/ϩ mice (Fig.  2). These results suggested the IRS-2 signal to not always be required for normal Pdx-1 expression.

The Effect of Hyperglycemia Induced by Aging or a High Fat Diet on Pdx-1 Expression in Irs2
Ϫ/Ϫ Murine ␤-Cells-Irs2 Ϫ/Ϫ mice progressively develop hyperglycemia starting at 10 weeks (11). To clarify the effect of aging or glucose toxicity on Pdx-1 expression, immunohistochemistry was performed on pancreata of 30-week-old diabetic Irs2 Ϫ/Ϫ mice. Pdx-1 protein was not reduced in the nuclei of ␤-cells from old Irs2 Ϫ/Ϫ mice (Fig. 5). Irs2 Ϫ/Ϫ mice on a high fat diet showed marked aggravation of glucose intolerance with exacerbation of obesity (Table I). Even in this state, the expression of Pdx-1 protein in ␤-cells, as well as those of downstream genes such as insulin and Glut2, did not show apparent reduction (Fig. 6, A and B).
Severe Diabetes Seen in Irs2 Ϫ/Ϫ Mice with a C57BL/6J ϫ 129Sv Mixed Background-We have maintained Irs2 Ϫ/Ϫ mice with a C57BL/6J or a C57BL/6J ϫ CBA hybrid background. We intercrossed our C57BL/6J background Irs2 Ϫ/Ϫ mice with mice having a C57BL/6J ϫ 129Sv hybrid background. Irs2 Ϫ/Ϫ mice with a C57BL/6J ϫ 129Sv hybrid background were divided in two groups based on blood glucose level; some Irs2 Ϫ/Ϫ mice showed mild hyperglycemia, as did those with only a C57BL/6J background, whereas other Irs2 Ϫ/Ϫ mice had very severe diabetes, dying due to dehydration with polyuria. Immunohistochemical studies revealed Pdx-1 protein to be generally undetectable as well as insulin in ␤-cells from these mice, whereas it was well preserved in mildly diabetic Irs2 Ϫ/Ϫ mice (Fig. 7). DISCUSSION The results of this study show Pdx-1 expression not to be down-regulated in ␤-cells from our Irs2 Ϫ/Ϫ mice with a C57BL/6J background through real time PCR, immunohistochemistry, and Western blot analysis, consistent with our previous report (11) showing increased glucose-induced insulin secretion and comparable insulin content in our Irs2 Ϫ/Ϫ murine islets. In addition, expression of genes lying upstream from Pdx-1, such as HNF3␤ and HNF1␣, as well as that of downstream target genes, including insulin, Glut2, and Nkx6.1, was well preserved in our Irs2 Ϫ/Ϫ murine ␤-cells.
Then what factors are involved in severely reduced Pdx-1 expression in ␤-cells from Irs2 Ϫ/Ϫ mice, as previously reported by another laboratory (37)? The involvement of glucose toxicity has been suggested. Their Irs2 Ϫ/Ϫ mice have been reported to show a wide range of fasting blood glucose levels (lower group, 99 Ϯ 4 mg/dl; higher group, 290 Ϯ 22 mg/dl) at 8 -10 weeks of age (9, 43), whereas our Irs2 Ϫ/Ϫ mice show comparatively lower (ϳ100 mg/dl) levels at 10 weeks of age (11). Hyperglycemia can down-regulate Pdx-1 expression (44,45), thereby impairing ␤-cell function and further aggravating hyperglycemia. However, our study revealed that, in 30-week-old diabetic Irs2 Ϫ/Ϫ mice, there was no decrease in Pdx-1 expression in ␤-cells. In  6. Pdx-1 expression is not down-regulated in islets from Irs2 ؊/؊ mice on a high fat diet for 5 weeks. A, representative sections of pancreata from Irs2 ϩ/ϩ and Irs2 Ϫ/Ϫ mice on a normal chow diet or a high fat diet, immunostained with antibodies against insulin, glucagon, somatostatin, pancreatic polypeptide, Pdx-1, and Glut2. Pancreata from four or five mice per group were examined. a-d, insulin is represented by brown staining, and non-␤-cells are represented by red staining with antibodies against glucagon, somatostatin, and pancreatic polypeptide. e-h, Pdx-1 is represented by brown staining. i-l, Glut2 is represented by red staining. B, Western blots of Pdx-1 in islets isolated from Irs2 ϩ/ϩ and Irs2 Ϫ/Ϫ male mice on normal chow diet or a high fat diet. For each group, islets from two mice were pooled, and lysates (15 g of protein) were subjected to Western blot analysis with antibodies against Pdx-1 and actin as an internal control. addition, even in Irs2 Ϫ/Ϫ mice on a high fat diet with blood glucose levels over 400 mg/dl, Pdx-1 expression was not downregulated. Thus, hyperglycemia itself may not be a major factor down-regulating Pdx-1 expression in ␤-cells from our Irs2 Ϫ/Ϫ mice.
Another difference between their Irs2 Ϫ/Ϫ mice and ours is the genetic background (C57BL/6J ϫ 129Sv versus C57BL/6J). Some previous reports have demonstrated that genetic backgrounds can affect the severity of phenotype in knockout mouse models (46 -48). We suspected that certain genetic 129Sv components might be involved in the Pdx-1 expression in Irs2 Ϫ/Ϫ mice, either directly or indirectly as modifier genes. When our Irs2 Ϫ/Ϫ mice were crossed with mice having a 129Sv background, a significant proportion of F 2 mice developed severe diabetes and died before several months of age, although others remained mildly diabetic and lived at least 1 year. When mildly diabetic F 2 mice were crossed with 129Sv, the proportion of severely diabetic Irs2 Ϫ/Ϫ mice was larger. Thus, certain genetic components in 129Sv mice apparently contribute to the development of severe diabetes (49). We found Pdx-1 expression to be reduced in ␤-cells from severely diabetic Irs2 Ϫ/Ϫ mice (Fig.  7). Our data strongly suggest that IRS-2 is not always required for the normal expression and function of Pdx-1, because disruption of IRS-2 did not affect the expression of Pdx-1 and its downstream target genes in our knockout mouse ␤-cells with a C57BL/6J background even when severe hyperglycemia was induced by a high fat diet. Cross-breeding experiments revealed that Pdx-1 reduction in Irs2 Ϫ/Ϫ mouse ␤-cells is a straindependent phenomenon. Kushner et al. (37) demonstrated that Pdx-1 expression is already reduced in their Irs2 Ϫ/Ϫ islets at birth, suggesting that Pdx-1 expression in ␤-cells is regulated via IRS-2 signaling in their mouse models. Considering their results and ours, when the genetic 129Sv components are present, IRS-2 might regulate the expression of Pdx-1. Another hypothesis is that certain 129Sv-related genetic elements may be involved in Pdx-1 reduction by enhancing the inhibitory effect of chronic hyperglycemia on the expression of Pdx-1, since Pdx-1 is a potential target of the glucose toxicity (44,50). This hypothesis might explain the mechanisms of age-related intense aggravation on ␤-cell dysfunction in their Irs2 Ϫ/Ϫ mice (9,10,37). Further study is anticipated to clarify this issue.
Then what common pathways are responsible for ␤-cell growth failure in their and our Irs2 Ϫ/Ϫ mice? ␤-cell-specific constitutively active Akt-1 transgenic mice reportedly have an increased ␤-cell mass (19,20) and suggest the involvement of IRS-2/Akt pathway in ␤-cell mass regulation (51). The Foxo1 pathway might also be involved in ␤-cell growth failure in Irs2 Ϫ/Ϫ mice. In addition to its role in metabolic regulation, Foxo1 might be involved in ␤-cell growth regulation as a downstream effector of IRS-2/Akt (52).
Our observations suggest that Pdx-1 reduction in Irs2 Ϫ/Ϫ murine ␤-cells is a strain-dependent event, and, at least with a C57BL/6J background, IRS-2 is not a major regulator of Pdx-1 expression in mouse ␤-cells. The mechanisms of ␤-cell growth failure, which is commonly observed in Irs2 Ϫ/Ϫ mice with any background, should be distinguished from insulin secretory dysfunction with Pdx-1 reduction.