Deficiency of the Tumor Promoter Gene wip1 Induces Insulin Resistance

Abstract

Diabetes is a growing health care issue, and prediabetes has been established as a risk factor for type 2 diabetes. Prediabetes is characterized by deregulated glucose control, and elucidating pathways which govern this process is critical. We have identified the wild-type (WT) p53-inducible phosphatase (WIP1) phosphatase as a regulator of glucose homeostasis. Initial characterization of insulin signaling in WIP1 knockout (WIP1^KO) murine embryo fibroblasts demonstrated reduced insulin-mediated Ak mouse transforming activation. In order to assess the role of WIP1 in glucose homeostasis, we performed metabolic analysis on mice on a low-fat chow diet (LFD) and high fat diet (HFD). We observed increased expression of proinflammatory cytokines in WIP1^KO murine embryo fibroblasts, and WIP1^KO mice fed a LFD and a HFD. WIP1^KO mice exhibited glucose intolerance and insulin intolerance on a LFD and HFD. However, the effects of WIP1 deficiency cause different metabolic defects in mice on a LFD and a HFD. WIP1^KO mice on a LFD develop hepatic insulin resistance, whereas this is not observed in HFD-fed mice. Mouse body weights and food consumption increase slightly over time in LFD-fed WT and WIP1^KO mice. Leptin levels are increased in LFD-fed WIP1^KO mice, compared with WT. In contrast, HFD-fed WIP1^KO mice are resistant to HFD-induced obesity, have decreased levels of food consumption, and decreased leptin levels compared with HFD-WT mice. WIP1 has been shown to regulate the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, loss of which leads to increased inflammation. We propose that this increased inflammation triggers insulin resistance in WIP1^KO mice on LFD and HFD.

Insulin resistance and obesity are risks for the development of diabetes. Identifying pathways that govern these processes is critical for the development of effective therapies. The WIP1 phosphatase is potentially linked to the control of metabolism through its negative regulation of ATM and tumor suppressor p53 (p53), which have roles in maintaining glucose homeostasis (1). The ATM protein, encoded by the Atm gene, is mutated in patients with ataxia telangiectasia, a recessive progeroid disease of early childhood, which includes an increased risk of insulin resistance and type 2 diabetes (2, 3). Our work has shown that ATM heterozygous mice on a chow diet become glucose intolerant at 6 months of age (1). In addition, mice deficient for ATM placed on an apolipoprotein E (ApoE)^−/− genetic background develop metabolic disease, which is characterized by insulin resistance, increased cholesterol and lipid levels, blood pressure, and atherosclerosis (4). ATM is activated by a number of stresses that have a direct impact on metabolism. A number of these cellular stresses, including atherosclerosis, trauma, hypoxia, and infections, are associated with p53 activation. ATM has been shown to directly phosphorylate and activate p53 on a site (Ser¹⁵) that regulates transcription activity (5–7). Many of the metabolic stresses that activate ATM also cause phosphorylation of p53 on the primary ATM site (Ser¹⁵ in humans, Ser¹⁸ in the mouse). To test whether the ATM pathway that regulates insulin resistance is mediated by p53 phosphorylation, we examined insulin sensitivity in mice with a germ-line mutation that replaces the p53 Ser¹⁸ (human Ser¹⁵) phosphorylation site with alanine. These p53^S18A mice exhibited defects in p53-mediated apoptosis and gene expression (see references 10 and 42 below), but unlike p53-null mice, p53^S18A mice developed tumors only at advanced age (1). p53^S18A mice developed increased metabolic stress, including severe defects in glucose homeostasis (1). The mice developed glucose intolerance and hepatic insulin resistance at 6 months of age. The p53^S18A mice had increased body weight and lipids and increased circulating levels of leptin, insulin, and proinflammatory cytokines TNFα and IL-6 compared with wild-type (WT) mice. Phosphorylation of p53Ser¹⁸ regulated expression of the antioxidants sestrin 2 and 3 in the liver, and hematopoietic zinc finger (a regulator of adipose tissue) in adipose tissue (1, 8). Increased oxidative stress was observed in murine embryo fibroblasts (MEFs) from p53^S18A mice. The glucose intolerance in p53^S18A mice was rescued by addition of an antioxidant in the diet. This suggested that increased oxidative stress was contributing to the defects in glucose homeostasis in p53^S18A mice. Further studies with the p44 activating allele of p53 confirmed the role of p53 in control of glucose homeostasis (8). These studies demonstrate that p53 phosphorylation on an ATM site is an important mechanism in the physiological regulation of glucose homeostasis and that regulation of oxidative stress contributes to this process.

We hypothesized that the WIP1 phosphatase, due to its regulation of the ATM-p53 pathway, would have a role in the control of glucose homeostasis. Wip1 (wt-p53-induced phosphatase) was identified in 1997 as a gene induced by ionizing radiation in a p53-dependent manner (9). The wip1 gene encodes a phosphatase that has substrate specificity to phosphorylated p-(S/T)Q amino acid motifs (10, 11). Substrates include tumor suppressors ATM (12), checkpoint kinase 2 (13), p53 (13), the p53-E3 ligase mdm2 (14), and the stress-responsive p38 MAPK (15). Thus, WIP1 participates in a regulatory feedback loop of the p53 pathway (12–14). Consistent with its role as a negative regulator of the ATM-p53 tumor suppressor pathway, wip1 is amplified in numerous tumors (16–18). Mice deficient for wip1 (WIP1 knockout [WIP1^KO] mice) are viable and exhibit defects in immunity, reproductive organs, cell cycle control, and display resistance to age-associated cancer (19–21). Genetic ablation of wip1 in mice also results in decreased tumorigenesis in cancer-prone models (22, 23). Furthermore, loss of wip1 results in enhanced phosphorylation of p53 (20) and ATM (12) in various mouse tissues.

We predicted that loss of WIP1 could lead to improved glucose homeostasis. Because WIP1 deficiency increases the activation of ATM and p53Ser¹⁸ in metabolically active tissues, increased activation in WIP1^KO mice could lead to improved glucose homeostasis. However, WIP1^KO mice also have increased inflammation, including expression of TNFα, IL-6, and C-reactive peptide (24). These cytokines are implicated in insulin resistance (25). It is not clear whether this induction of inflammation in WIP1^KO mice would override the potential benefits of increased ATM and p53 activity. Indeed, a recent study has reported that loss of WIP1 in an ApoE^−/− background leads to increased atherosclerosis (26). However, the impact of loss of wip1 in glucose tolerance and insulin resistance has not been examined. Thus, to establish the role of WIP1 in glucose homeostasis, we have performed a metabolic analysis of animals on a low-fat chow diet (LFD) and high-fat diet (HFD). The expression of inflammatory cytokines, such as TNFα and IL-6, was increased in a tissue-specific manner in WIP1^KO-deficient animals. Interestingly, WIP1^KO mice exhibited glucose intolerance and insulin intolerance on the LFD and HFD. However, WIP1^KO mice also exhibited different metabolic phenotypes. WIP1^KO mice on the LFD exhibited hepatic insulin resistance, increased fat mass, and increased circulating levels of insulin and leptin. In contrast, HFD-fed WIP1^KO mice exhibited decreased circulating levels of leptin and insulin compared with WT and decreased food consumption and decreased body mass. Thus, WIP1 has differential roles in the response to LFD and HFD. Overall, these results implicate WIP1 as a regulator of glucose homeostasis in vivo.

Research Design and Methods

Mouse strains and diet information

The methods employed for the generation and genotyping of WIP1^KO have been reported previously (19). Wip1^−/+ mice were intercrossed to obtain wip1^−/− (WIP1^KO) and WT mice. Cohorts of male mice on a mixed 129/Sv x C57BL/6 genetic background were established for analysis. Animals were placed on a chow diet or a HFD (Bioserv F3282, 60% fat calories; Bioserv). The mice were housed in specific pathogen-free facilities accredited by the American Association for Laboratory Animal Care. The Institutional Animal Care and Use Committees of the University of Massachusetts Medical School approved all studies using animals.

Glucose tolerance test (GTT) and insulin tolerance test (ITT)

Mice were examined using a GTT and an ITT with methods described previously (27). Briefly, mice were fasted (GTT) or not fasted (ITT) overnight and challenged by ip administration of glucose (1 g/kg body weight, IMS) or insulin (0.75 U/kg body weight). Blood glucose was measured with an Ascenzia Breeze 2 glucometer (Bayer). Serum insulin and leptin levels were measured by ELISA multiplex (MAP KIT MMHMAG-44k, Luminex 200; EMD Millipore).

Hyperinsulinemic-euglycemic clamp studies

The clamp studies were performed at the University of Massachusetts Obesity Mouse Phenotyping Center. Whole-body fat and lean mass were noninvasively measured using ¹H-MRS (Echo Medical Systems). After an overnight fast, a 2-hour hyperinsulinemic-euglycemic clamp was conducted in conscious mice with a primed and continuous infusion of human insulin (150 mU/kg body weight priming followed by 2.5 mU/kg·min; Humulin; Eli Lilly), and 20% glucose was infused at variable rates to maintain euglycemia (28). Whole-body glucose turnover was assessed with a continuous infusion of [3-³H]glucose (PerkinElmer), and 2-deoxy-D-[1-¹⁴C]glucose (PerkinElmer) was administered as a bolus (10 μCi) at 75 minutes after the start of clamp to measure insulin-stimulated glucose uptake in individual organs (28).

Analysis of tissue sections

Histology was performed using tissue fixed in 10% formalin for 24 hours, dehydrated, and embedded in paraffin. Sections (7 μm) were cut, stained using hematoxylin and eosin (American Master Tech Scientific). Sections were viewed through an Olympus BX-41 microscope and examined by a board-certified veterinary pathologist. Pictures of slides were taken with a Media Cybernetics–Evolution MP 5.0 camera. Relative size of adipocytes was determined using a ×20 digital histology figure using Canvas software and comparing the longest diameter of each cell, measured in millimeters as arbitrary units. A total of 60 cells were measured on chow diet and 22 cells measured on HFD per animal. Estimation of relative islet size was determined using a ×4 digital histology figure in the Canvas program and comparing the longest diameter of each islet and measured in millimeters. Five animals were analyzed per genotype and treatment. A total of 15 islets was measured on chow diet and 40 islets measured on HFD. This measurement did not include sufficient data for determination of islet mass.

Immunoblot analysis

Tissues extracts were prepared as described (1) and were examined by immunoblot analysis using antibodies to mouse Ak mouse transforming (AKT), phospho-Ser⁴⁷³ AKT, and phospho-Thr³⁰⁸ AKT (Cell Signaling) and analyzed on the Fuji Film LAS 4000. Readings of band intensity of the phospho-antibodies were determined and normalized for background and levels of AKT. Three separate gels were analyzed per experiment.

RNA preparation and analysis

RNA was prepared as described (1). The relative expression of mRNA was examined by quantitative PCR analysis. cDNA was prepared using Bio-Rad iScript with random hexamers and 0.5–1 μg of RNA per tissue. Quantitative real-time PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems 7500 Fast real-time system. The primer sequences for the murine genes were: wip1, 5′-CAGAAAGGCTTCACCTCGTC-3′ and 5′- CACCTCCACAGCTCTCACAA-3′ (29); IL-6, 5′-AAGAGACTTCCATCCAGTTGC-3′ and 5′-CTC CGA CTT GTGAAG TGG TAT-3′; TNFα, 5′-ACAGAAAGCATGATCCGCG-3′ and 5-CTGGGCCATAGAACTGATG-3′; and Gapdh, 5′-CTTCACCACCATGGAGAAGGC-3′ and 5′-GGCATGGACTGTGGTCAT-3′). All samples were examined in triplicate and values were normalized for baseline expression and for expression of Gapdh. Calculations of values were made using the ΔΔ cycle threshold method. Statistical significance was calculated using cycle threshold values.

Tissue culture

Primary MEFs were prepared from embryonic day 14 WT and WIP1^KO embryos (30). Briefly, embryos were removed at the embryonic day 14. Embryos were mechanically disrupted and digested with trypsin for 10 minutes. Samples were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen). Adherent fibroblasts were frozen at passage 0 or used in experiments. All studies were performed with MEFs at early passage number (passage 3–5).

Data analysis

Statistically significant differences (P < .05) between groups were examined using the 2-tailed Student's t test. Microsoft Excel was used for statistical calculations.

Results

Defective insulin signaling in WIP1^KO MEFs

Our previous published data demonstrated a crucial role for the ATM-p53 pathway in the maintenance of glucose homeostasis (1, 8). We hypothesized that WIP1 could regulate glucose homeostasis based on its ability to negatively regulate the ATM-p53 axis. However, because WIP1^KO MEFs also exhibit increased inflammation, the role of WIP1 to regulate insulin sensitivity was unclear. In support of previous reports that WIP1 loss leads to increased inflammation (19–21), the expression of IL-6 and TNFα mRNAs was markedly increased in WIP1^KO MEFs compared with WT MEFs (Figure 1A). It is known that increased expression of these proinflammatory cytokines can lead to insulin resistance. To test the effects of WIP1 deficiency on insulin sensitivity, we performed a time course of insulin treatment on WT and WIP1^KO MEFs. The cells were treated with insulin and AKT phosphorylation levels were examined (Figure 1B). Although treatment of WT MEFs lead to a clear increase in AKT phosphorylation at both Thr³⁰⁸ and Ser⁴⁷³, loss of WIP1 significantly mitigated this response (Figure 1B). This result was surprising based on our hypothesis that increased ATM and p53 activation in WIP1^KO mice would lead to improved glucose homeostasis. In addition, p38, another WIP1 target, is activated in WIP1^KO MEFs. Thus, these effects of loss of wip1 are not likely mediated by increased p38 signaling. In fact, activation of p38 has been shown to help maintain glucose homeostasis (31). In support that increased p38 activation did not mediate the effects of WIP1, pretreatment of cells with the p38 kinase inhibitor BIRB796 (32) did not lead to restoration of AKT activation in WIP1^KO MEFs (data not shown). Thus, increased activation of classic targets of WIP1 in WIP1^KO tissues are not likely to override the decreased insulin signaling due to deficiency of WIP1.

Figure 1.

Defects in insulin signaling in MEFs from WIP1^KO mice. A, The expression of TNFα, IL-6 and Gapdh mRNA was measured by quantitative real-time PCR analysis of MEFs from WT and WIP1^KO mice. The amount of Gapdh mRNA in each sample was used to calculate relative mRNA expression (mean ± SEM; n = 3). Statistically significant differences between WT and WIP1^KO mice are indicated (*, P < .05). B, Representative Western blotting of AKT activation in WT and WIP1^KO MEFs. Insulin treatment time course in WT MEFs (left panel) and WIP1^KO MEFs (right panel). Cells were starved for 18 hours and treated with 10nM insulin for the indicated times. Protein levels of AKT, AKT-pThr³⁰⁸, and AKT-pSer⁴⁷³ were examined by immunoblot. C, The expression of wip1 and Gapdh mRNA were measured by quantitative real-time PCR analysis. Wip1 expression in liver, WAT, and muscle from WT and WIP1^KO mice were compared. The amount of Gapdh mRNA in each sample was used to calculate relative mRNA expression (mean ± SEM; n = 3). Statistically significant differences between WT and WIP1^KO mice are indicated (*, P < .05).

Defective basal glucose homeostasis and insulin resistance in WIP1^KO mice

Decreased tissue insulin sensitivity and increased inflammation can lead to disruption of glucose homeostasis in vivo. We therefore examined glucose homeostasis in WIP1^KO and WT animals on a LFD. WIP1^KO mice exhibited glucose intolerance at 21 weeks of age on a chow diet (Figure 2A). To examine the contribution of resistance of peripheral tissues to insulin action, an ip ITT was performed. WIP1^KO mice exhibited elevated glucose levels in response to insulin, suggesting peripheral insulin resistance (Figure 2B). The insulin resistance was not due to increased overall body mass, because animals exhibited the same average weight from 8 to 24 weeks (Figure 2C). The WIP1^KO animals consumed similar amounts of food per week per animal (Figure 2D). However, the glucose intolerance exhibited by the mice during ip glucose stimulation could be due to either reduced insulin release into circulation and/or the resistance of peripheral tissues to the action of insulin. Thus, it is possible that defects in insulin release also contribute to the insulin resistance.

Figure 2.

Loss of WIP1 leads to insulin resistance and has no effect on weight gain under chow diet. WT and WIP1^KO mice were maintained on a standard LFD and fed ad libitum until 24 weeks of age. A and B, Black circles represent WT mice and black squares represent WIP1^KO mice. A, GTT on 21-week-old mice (5-months-old). Mice fasted overnight were treated with glucose (1 g/kg) by ip injection. Blood glucose concentration was measured at the indicated times (mean ± SEM; n = 20). B, ITT on 22-week-old mice (over 5-months-old). Mice were treated with insulin (0.75 U/kg) by ip injection. Blood glucose was measured at the indicated times (mean ± SEM; n = 20). C and D, Filled bars represent WT mice and open bars represent WIP1^KO mice. C, Body weight was measured from 8 to 24 weeks of age (mean ± SEM; n = 20). Arrows show the time of the GTT and ITT in A and B. D, Food intake was measured weekly. An average of weekly food consumption from 11 to 22 weeks was calculated (mean ± SEM; n = 20). A–D, Statistically significant differences are indicated with an asterisk (P < .05).

Further support for defects in insulin sensitivity was detected by insulin-mediated AKT activation in peripheral tissues. Examination of AKT activation determined that there was significantly reduced AKT phosphorylation in tissue from liver but not white adipose tissue (WAT) (Figure 3A, left and right panels, respectively). We observed decreased AKT phosphorylation in liver, but not WAT, indicating that hepatic loss of AKT activation is likely contributing to the glucose intolerance and insulin resistance observed in the animals.

Figure 3.

WIP1^KO mice exhibit reduced hepatic AKT activation and increased inflammation. WT and WIP1^KO mice were maintained on a standard LFD and fed ad libitum. A, Mice at 24 weeks of age were examined for insulin-mediated AKT activation in the liver (left panel) and WAT (right panel). The mice were fasted overnight and treated without and with insulin (1.5 U/kg body mass) by ip injection (30 minutes). Extracts prepared from the liver of WT and WIP1^KO mice. Protein levels of AKT, AKT-pThr³⁰⁸, and AKT-pSer⁴⁷³ were examined by immunoblot. B and C, Mice at 24 weeks of age were euthanized and tissues removed for expression analysis. The expression of TNFa, IL-6, and Gapdh mRNA was measured by quantitative real-time PCR analysis of liver (A) and WAT (B) of WT mice compared with WIP1^KO mice. The amount of Gapdh mRNA in each sample was used to calculate relative mRNA expression (mean ± SEM; n = 3). D, Levels of circulating insulin were determined on blood taken from WT and WIP1^KO mice at 8 and 20 weeks of age (mean ± SEM; n = 10). E, Measurement of leptin serum levels in blood taken from 8- and 20-week-old animals (mean ± SEM; n = 10). B–D, Statistically significant differences between WT and WIP1^KO mice are indicated (*, P < .05).

To further elucidate a mechanistic role for the glucose intolerance and insulin resistance, we examined mRNA expression of proinflammatory cytokines on animals at 24 weeks of age. Both TNFα and IL-6 were significantly increased in the liver of WIP1^KO mice compared with WT mice (Figure 3B). However, there was no difference in levels of TNFα in WAT, although IL-6 was significantly elevated (Figure 3C). We measured additional metabolic parameters, which can contribute to defects in glucose homeostasis, including circulating insulin and leptin levels. The serum levels of insulin and leptin were taken at a baseline of 8 weeks of age (Figure 3, D and E). The baseline levels of insulin in young animals (8-week-old) were similar in both genotypes. The levels were again compared on aged animals (20-week-old) on a LFD (Figure 3, D and E). The insulin levels in WIP1^KO animals were slightly increased but not significantly (Figure 3D).

We examined circulating serum levels of leptin (Figure 3E). At 8 weeks of age, LFD-fed WT mice exhibited low levels of leptin, consistent with low body weight (Figure 2C). At 8 weeks of age, LFD-fed WIP1^KO animals exhibited higher levels of leptin than WT mice, but this difference was not significant (Figure 3E). Leptin levels in WT mice increased only slightly from 8 to 20 weeks of age (Figure 3E). In contrast, leptin levels in WIP1^KO mice increased significantly from 8 to 20 weeks of age and were significantly greater than WT leptin levels (Figure 3E). Importantly, the increase in leptin in W1P1^KO animals (Figure 3E, 20 weeks) was correlated with normal food consumption in WIP1^KO animals (Figure 2D). Thus, the increased leptin levels did not lead to decreased food consumption in WIP1^KO mice, suggesting a degree of leptin resistance in the WIP1^KO mice.

To further test the hypothesis that loss of wip1 causes insulin resistance, we performed a hyperinsulinemic-euglycemic clamp study in conscious mice. The clamp analysis was performed on a cohort of 10 mice at 24 weeks of age. No significant difference in body weight was observed at the time of the clamp analysis (Figure 4A). Steady-state glucose infusion rate was significantly lower in WIP1^KO mice as compared with WT mice (Figure 4B). However, insulin-stimulated whole-body glucose turnover and glycolysis did not differ significantly between groups (Figure 4, C and D). Basal glucose and clamp glucose levels were similar during the experiment (Figure 4, E and F). The basal hepatic glucose production (HGP) rates were similar in WT and WIP1^KO mice (Figure 4G). However, insulin-mediated suppression of HGP was blunted, resulting in significantly elevated clamp HGP rates in WIP1^KO mice, indicating hepatic insulin resistance in these mice (Figure 4H). Although there was no difference in lean mass (Figure 4I), whole-body fat mass was increased in WIP1^KO mice on a LFD (Figure 4J). The increased fat mass correlated with increased leptin levels in WIP1^KO mice observed at 24 weeks (Figure 3E).

Figure 4.

WIP1^KO mice on a chow diet exhibit hepatic insulin resistance. WT and WIP1^KO mice were maintained on a standard LFD and analyzed at 24 weeks of age. A, Body weight of animals at time of hyperinsulinemia-euglycemic clamp study. B, Steady-state glucose infusion rates to maintain euglycemia during the clamp. C, Whole-body glycolysis. D, Insulin-stimulated whole-body glucose turnover. E, Basal blood glucose concentration. F, Blood glucose concentration during the hyperinsulinemia-euglycemic clamp (mean ± SEM; n = 7–8). G, Basal HGP. H, Insulin-mediated suppression of HGP. Lean mass (I) and fat mass (J) measurements. Statistically significant differences are indicated with an asterisk (*, P < .05). A–J, The data presented are the mean ± SEM (n = 7–8).

Histology on tissues was performed at the end of the experiment, when the animals were 24-weeks-old. Histological analysis revealed no differences in the morphology of the liver in WIP1^KO mice (Figure 5B) compared with livers of WT mice (Figure 5A). The livers also had similar weights upon necropsy (Figure 5C). WAT had similar morphology in WT and WIP1^KO mice (Figure 5, D and E), as well as relative cell size (Figure 5F). The morphology of islets were similar in WT and WIP1^KO mice (Figure 5, G and H). The relative islet size was also similar (Figure 5I).

Figure 5.

Tissues from aged WT and WIP1^KO mice fed ad libitum exhibit similar histology. WT and WIP1^KO mice were fed ad libitum with LFD diet until 24 weeks. Liver, WAT, and pancreas were immersion fixed and embedded in paraffin. Histological sections were stained with H&E. Photomicrographs depict liver (A and B), WAT (D and E), and pancreas (G and H) from WT (A, D, and G) and WIP1^KO mice (B, E, and H). Representative micrographs from WT (right panels) and WIP1^KO (left panels) tissues: WT and WIP1^KO mice display normal hepatocyte structure (×20) (A and B). C, Livers from chow-fed animals had similar weights upon necropsy. The data presented are the mean ± SEM (n = 8). Statistically significant differences are indicated with an asterisk (*, P < .05). D and E, Adipocyte structure in chow-fed animals (×20). F, WAT from WT and WIP1^KO mice have similar cell size. The data presented are the mean ± SEM (n = 60 cells). Statistically significant differences are indicated with an asterisk (*, P < .05). G and H, WT and WIP1^KO mice display similar islets (×20). I, WT and WIP1^KO mice contain similar islets size. The data presented are the mean ± SEM (n = 15 islets, 3 animals per genotype, 5 animals per genotype). Statistically significant differences are indicated with an asterisk (*, P < .05).

HFD induces metabolic defects in WIP1^KO mice

The defects in insulin signaling in MEFs and insulin resistance observed in WIP1^KO animals indicated that loss of WIP1 could be important in the development of insulin resistance in response to HFD. To test this hypothesis, animals were placed on a HFD. Figure 6A shows a schematic diagram of the HFD experiment, including the time when the various experiments were performed. The duration of the HFD was for 16 weeks, until the mice were 24 weeks of age. A cohort of mice was removed from the group after 18 weeks and kept on HFD until clamp analysis. WIP1^KO mice exhibited glucose intolerance (Figure 6B) and insulin intolerance (Figure 6C). We measured weight and food consumption during the HFD (Figure 6, D and E). The weight of WT and WIP1^KO mice increased with time on the HFD, as expected (Figure 6D). Interestingly, the WIP1^KO animals gained less body mass compared with WT animals (Figure 6D). In addition, we observed a decrease in average weekly food consumption per animal in WIP1^KO mice compared with WT mice (Figure 6E). The smaller body mass of the animals could account for the improved insulin tolerance in HFD-fed WIP1^KO mice compared with LFD-fed WIP1^KO mice (compare Figure 2B and Figure 6C). We also analyzed food intake adjusted for body weight (food to weight ratio) to determine whether there was a difference in the efficiency in weight gained per food consumed. We found that the average food to weight ratio from 10 to 13 weeks, where there is no weight difference (see Figure 6D) was 0.68 and 0.71 for WT and WIP1^KO mice, respectively. The average food to weight ratio for weeks 14–18 was 0.49 and 0.32 for WT and WIP1^KO mice, respectively. This suggests that additional factors are likely contributing to the improved ability of WIP1^KO mice to gain weight for the food consumed.

Figure 6.

Defects in glucose homeostasis in WIP1^KO mice on a HFD. WT and WIP1^KO mice were maintained on a HFD starting at 8 weeks of age. A, Schematic diagram of HFD experiment. B, GTT performed on animals at 16 weeks of age. Mice fasted overnight were treated with glucose (1 g/kg) by ip injection. Blood glucose concentration was measured at the indicated times (mean ± SEM; n = 20). C, ITT on animals in A at 18 weeks of age. Mice were treated with insulin (0.75 U/kg) by ip injection. Blood glucose was measured at the indicated times (mean ± SEM; n = 20). D, Mouse weights were measured weekly. Average mouse body weight from 8 to 22 weeks of age of HFD-fed WT and WIP1^KO mice. E, Food intake was measured weekly. Average weekly food consumption per mouse from 10 to 18 weeks is shown. F, Measurement of circulating leptin levels in blood taken from 20-week-old HFD-fed mice compared with 20-week-old LFD-fed mice (mean ± SEM; n = 10). B–F, Statistically significant differences between WT and WIP1^KO mice are indicated (*, P < .05).

We examined circulating levels of leptin in the HFD animals at 20 weeks of age, after 12 weeks on a HFD (Figure 6F). In HFD-fed mice, leptin levels were dramatically elevated compared with LFD-fed animals at 20 weeks. Interestingly, the leptin levels were significantly lower in HFD-fed WIP1^KO animals compared with HFD-fed WT animals. Thus, despite decreased leptin levels (Figure 6F), the WIP1^KO mice consumed less food than WT mice (Figure 6E). In addition, a decrease in weight and food is usually seen when leptin levels are high. However, the WIP1^KO mice had effects on food consumption which would normally be seen with increased levels of leptin, suggesting that WIP1^KO mice on a HFD were more sensitive to the effects of leptin. This was in contrast to the leptin resistance observed under LFD (Figures 2D and 3E).

To examine insulin sensitivity, we determined insulin-mediated activation of AKT by immunoblot analysis. The analysis was done at the end of the HFD experiment, when the mice were 24-weeks-old (Figure 7A). The 2 panels in Figure 7A are representative immunoblots of insulin-mediated AKT activation. There was very little activation of AKT in the liver of WT and WIP1^KO animals, consistent with the insulin resistance seen in animals placed on a HFD (Figure 7A, left panel). The induction of AKT was more robust in the WAT (Figure 7A, right panel). Importantly, there was no difference in AKT activation in WT or WIP1^KO tissues from animals placed on a HFD.

Figure 7.

Increased inflammation and insulin levels in HFD-fed WIP1^KO mice. WT and WIP1^KO mice were maintained on a HFD starting at 8 weeks of age. A, Mice at 24 weeks of age were examined for insulin-mediated AKT activation in the liver (left panel) and WAT (right panel). The mice were fasted overnight and treated without and with insulin (1.5 U/kg body mass) by ip injection (30 minutes). Extracts prepared from the liver of WT and WIP1^KO mice. Protein levels of AKT, AKT-pThr³⁰⁸, and AKT-pSer⁴⁷³ were examined by immunoblot. B and C, The expression of TNFα, IL-6, and Gapdh mRNA was measured by quantitative real-time PCR analysis of liver (B) and WAT (C) of WT mice compared with WIP1^KO mice. The amount of Gapdh mRNA in each sample was used to calculate relative mRNA expression (mean ± SEM; n = 3). D, Measurement of insulin serum levels in 8- and 20-week-old animals (mean ± SEM; n = 10). B–D, Statistically significant differences between WT and WIP1^KO mice are indicated (*, P < .05).

Tissues were removed for expression analysis at the end of the HFD experiment. To test whether increased inflammation was observed in WIP1^KO animals placed on a HFD, we examined the mRNA expression of proinflammatory cytokines TNFα and IL-6. HFD-fed WIP1^KO animals had significantly increased hepatic TNFα expression compared with WT (Figure 7B). However, the liver expression of IL-6 was not elevated in HFD WIP1^KO mice (compare Figure 7B and Figure 3B). In addition, HFD WT and WIP1^KO mice had similar expression levels of TNFα and IL-6 in WAT (Figure 7C). We examined circulating levels of insulin in the HFD animals at 20 weeks of age, after 12 weeks on a HFD (Figure 7D). Insulin levels were significantly lower in HFD WIP1^KO mice.

To determine the state of insulin sensitivity, we performed a hyperinsulinemic-euglycemic clamp study in conscious mice during the HFD. We performed the clamp analysis on a cohort of similar weight-matched animals that exhibited glucose intolerance, based on our observation that insulin resistant chow-fed WIP1^KO animals had similar weight to WT animals (Figure 3). The animals had similar weights at the time of the clamp analysis (Figure 8A). There was no difference in glucose infusion rate in WIP1^KO mice (Figure 8B). However, whole-body glycolysis was significantly greater in WIP1^KO mice (Figure 8C), suggesting a degree of insulin resistance. Glucose turnover, basal glucose, and clamp glucose in the WT and WIP1^KO mice during the clamp were similar (Figure 8, D–F). The basal hepatic glucose (HGP) and clamp HGP in WT and WIP1^KO mice were similar (Figure 8, G and H). There was no difference in lean mass or fat mass during high-fat feeding (data not shown). The overall clamp analysis revealed mild insulin resistance in WIP1^KO mice. This is consistent with the observation of very little induction of AKT in the liver of WT and WIP1^KO mice (Figure 7A).

Figure 8.

Clamp analysis in WIP1^KO animals in response to long-term HFD. WT and WIP1^KO mice were maintained on a HFD for 16 weeks and analyzed by hyperinsulinemia-euglycemic clamp. A, Body weight of animals at time of hyperinsulinemia-euglycemic clamp study. B, Steady-state glucose infusion rates to maintain euglycemia during the clamp. C, Whole-body glycolysis. D, Insulin-stimulated whole-body glucose turnover. E, Basal blood glucose concentration. F, Blood glucose concentration during the hyperinsulinemia-euglycemic clamp. Basal HGP (G) and insulin-stimulated HGP (H) during the hyperinsulinemia-euglycemic clamp. Statistically significant differences are indicated with an asterisk (*, P < .05). The data presented are the mean ± SEM (n = 7–8).

At 24 weeks of age, the HFD feeding was concluded, the animals were euthanized, and organs were analyzed by histology. The liver of HFD-fed WT mice exhibited steatosis (Figure 9A), compared with the liver of LFD-fed WT mice (Figure 5A). The HFD-fed WIP1^KO animals also exhibited increased lipid invasion in the liver, with mostly smaller sized vacuoles or microsteatosis (Figure 9B). Interestingly, 50% of HFD WIP1^KO animals exhibited microsteatosis, whereas only 12% of HFD WT mice exhibited microsteatosis. However, the size of the livers from the HFD mice were similar (Figure 9C). The WAT exhibited similar morphology and relative cell size (Figure 9, D–F). The islets from HFD-fed WT mice had enlarged islets, compared with LFD-fed mice (compare Figure 5G and Figure 9G). The islets in the HFD-fed WIP1^KO mice (Figure 9H) were smaller than HFD-fed WT mice (Figure 9I).

Figure 9.

Histology of WT and WIP1^KO animals on HFD. WT and WIP1^KO mice were placed on HFD for from 8 to 24 weeks of age. H&E-stained sections were prepared and analyzed. Representative photomicrographs from liver (A and B), WAT (D and E), and pancreas (G and H) from WT (A, D, and G) and WIP1^KO (B, E, and H) mice. All pictures are at ×20. A, HFD induces steatosis in livers from WT mice. B, WIP1^KO mice exhibit greater microsteatosis. C, Livers from chow-fed animals had similar weights upon necropsy. The data presented are the mean ± SEM (n = 8). Statistically significant differences are indicated with an asterisk (*, P < .05). D and E, Adipocyte structure is enlarged in WT (D) and WIP1^KO mice on a HFD (F) WAT from WT and WIP1^KO mice have similar cell size. The data presented are the mean ± SEM (n = 22 cells). Statistically significant differences are indicated with an asterisk (*, P < .05). G, A representative enlarged islet from HFD-fed WT mice. H, A representative islet from HFD-fed WIP1^KO mice. I, Relative islet size depicting increased islet diameter. The data presented are the mean ± SEM (n = 40). Statistically significant differences are indicated with an asterisk (*, P < .05).

Our studies demonstrate a role for WIP1 in maintaining glucose homeostasis. WIP1^KO mice on a LFD and HFD developed glucose intolerance and insulin intolerance. Interestingly, WIP1^KO mice displayed different phenotypes on the LFD and HFD. The LFD WIP1^KO mice exhibited hepatic insulin resistance. The development of insulin resistance was not due to weight gain or increased feeding and was correlated with increased expression of proinflammatory cytokines. LFD WIP1^KO mice expressed increased levels of circulating leptin. However, the mice had similar food consumption compared with LFD WT mice. WIP1^KO mice on the HFD exhibited more severe insulin intolerance, yet did not exhibit hepatic insulin resistance. HFD WIP1^KO animals had decreased weight gain and food consumption over time compared with HFD-fed WT mice. The HFD WIP1^KO mice exhibited significantly lower circulating levels of insulin and leptin. These studies all support a role of WIP1 in maintaining glucose homeostasis.

Discussion

The WIP1 protein is a member of the ATM-p53 axis in control of DNA damage control and tumor suppression. WIP1 has been shown to negatively regulate both ATM (12) and p53 (13). WIP1 also dephosphorylates the p38 stress kinase (15). We have previously demonstrated that the ATM-p53 pathway is required for glucose homeostasis (1). The negative role of WIP1 on the ATM-p53 pathway lead us to hypothesize that WIP1 would be a mediator of insulin resistance in vivo. Therefore, we predicted that loss of wip1 would lead to improved glucose homeostasis. We have previously shown that an activated allele of p53 (p44) leads to improved glucose homeostasis (8). Furthermore, p38MAPK, also negatively regulated by WIP1, has a protective role in diet-induced obesity and insulin resistance (31). Preliminary studies in knockout MEFs indicated that insulin signaling was decreased in WIP1^KO cells compared with WT cells (Figure 1). WIP1^KO MEFs exhibited reduced activation of AKT in response to insulin. In addition, expression of the proinflammatory cytokines TNFα and IL-6 were significantly increased (Figure 1). Thus, to further examine the contribution of WIP1 to glucose homeostasis, we performed metabolic analysis of WIP1^KO mice (19). Surprisingly, animals on a LFD exhibited glucose intolerance and decreased insulin sensitivity (Figure 2). This was correlated with decreased insulin-mediated AKT activation in liver tissue (Figure 3). There was tissue-specific regulation of proinflammatory cytokines. TNF and IL-6 were elevated in the liver of WIP1^KO mice. However, only IL-6 was elevated in the WAT. Clamp analysis of LFD WIP1^KO animals indicated hepatic insulin resistance and increased fat mass (Figure 4).

The weight and food consumption of WT and WIP1^KO mice on LFD were similar. Insulin levels were also similar, despite insulin intolerance. Surprisingly, leptin levels were higher in the WIP1^KO mice. The higher levels of leptin could be due to the increased fat mass, because leptin levels parallel fat mass. However, an increase in leptin would be expected to lead to a decrease in food consumption. However, the animals had similar food consumption, despite increased leptin. One possible explanation could be that the WIP^KO mice are not responding to leptin and had developed a certain degree of leptin resistance. However, a direct measurement of leptin activity would be required to test this.

We also placed the WIP^KO mice on a HFD to determine the effects of loss of wip1 on obesity-mediated insulin resistance. The metabolic profile was different in WIP1^KO mice in response to HFD compared with LFD. The HFD WIP1^KO mice exhibited glucose intolerance and more severe insulin intolerance, yet they did not exhibit hepatic insulin resistance. One caveat of the clamp experiment on the HFD was that it was done 6–8 weeks after the GTT and ITT. Therefore, it is possible that extended time on the HFD made the WIP1^KO animals more similar to WT mice in insulin resistance. In addition, the mice that were used for clamp analysis had no significant difference in body weight.

Although HFD increased the body weight over time in WT and WIP1^KO mice as expected, WIP1^KO mice developed significantly reduced body weight compared with WT mice. Thus, WIP1^KO mice were protected from HFD-induced obesity. Indeed, this was recently reported in another study (26). In addition to body weight, we report that food consumption was also reduced in WIP^KO mice on the HFD. Leptin levels increased in response to HFD in WT and WIP1^KO mice compared with LFD animals. However, WIP1^KO mice had significantly less leptin than WT mice on the HFD. Decreased leptin would be expected to lead to an increase in food consumption. Thus, for the decreased level of leptin, the WIP1^KO animals were more sensitive to leptin. The food consumption and weight were similar, suggesting increased leptin sensitivity. We propose that increased leptin sensitivity can contribute to the decreased food consumption in WIP1^KO mice. In addition, we analyzed food intake adjusted for body weight (food to weight ratio). The weight and food consumption are decreased from 14 to 18 weeks in the HFD-fed WIP1^KO mice. The average food to weight ratio for these weeks was 0.49 and 0.32 for WT and WIP1^KO mice, respectively, suggesting that the WIP1^KO mice have additional factors affecting weight gain. For instance, it is possible that other regulators of hypothalamic control, such as ghrelin, are also regulated by WIP1. Perturbations in metabolic rate could also contribute to the defects in our study. Indeed, it was recently reported that loss of wip1 in an ApoE^−/− background leads to increased metabolic rate (26). Interestingly, WIP1^KO mice were protected from HFD-induced increases in insulin levels. In addition, HFD-fed WT mice appeared to have more islet hyperplasia.

There are similar defects in metabolism in the LFD and HFD WIP1^KO mice, which can contribute to insulin resistance. For instance, the proinflammatory cytokine TNFα was elevated in the liver in LFD and HFD WIP1^KO mice. We have also not ruled out defects of glucose-mediated insulin release, although insulin levels were differentially regulated during LFD and HFD. However, there are also clear differences in metabolic defects in LFD and HFD WIP1^KO mice. In LFD mice, WIP^KO mice exhibited a degree of leptin resistance. This could contribute to the central-mediated energy expenditure. We have not ruled out differences in metabolic rates in the LFD mice. The HFD WIP1^KO mice appear to have an increased leptin sensitivity.

The glucose and insulin intolerance of WIP1^KO mice under LFD and HFD diet are unexpected given the protective role both ATM and p53 have been shown to play in glucose homeostasis (1). WIP1^KO mice have been shown to have increased ATM and p53 activation, which have been shown to be important for glucose homeostasis. In addition, we have shown that increased p53 signaling improves glucose homeostasis (8). However, other groups have reported a role of p53 in promoting insulin resistance (33, 34). Our data suggest that the WIP1 pathway is protecting against glucose intolerance in normal aging and diet-induced insulin resistance. This role is likely to be dominant over the potential positive role of the increased activation of the ATM-p53 pathway due to wip1 deficiency. WIP1^KO mice exhibit increased inflammation and immune response; it is possible that this is mediating the insulin resistance observed in WIP1^KO animals. However, other factors such as regulation of metabolic rate can be contributing to insulin resistance.

A negative role for metabolism has been demonstrated for WIP1 in the control of atherosclerosis. Recently, it was published that loss of wip1 leads to improved atherosclerosis (26). In this report, WIP1^KO mice in an ApoE^−/− background exhibited fewer atherosclerosis lesions. In accordance with our data, they observed protection from HFD-induced weight gain in WIP1^KO/ApoE^−/− mice. Thus, our data support the observation that WIP1^KO mice are resistant to diet-induced obesity. However, the authors reported that food intake was unchanged in WIP1^KO animals. Our observations show that food intake is significantly reduced in HFD-fed WIP1^KO animals compared with WT animals. It is possible that genetic difference due to the different mouse strains used (mixed 129/6 and B6) could contribute to these difference. The authors reported an improvement in the metabolic rate of the WIP1^KO mice. Examination of food to weight ratios suggests factors additional to caloric intake can be affecting the reduced weight. In addition, because these WIP1^KO mice are whole-body knockouts, it is possible that WIP1 plays an as yet unidentified role in regulation of leptin-sensitive neurons.

Together, these data indicate that the WIP1 phosphatase functions to maintain insulin sensitivity and glucose homeostasis. This role is likely to be dominant over any protective effects from increased ATM and p53 (12, 13). Importantly, our studies characterize a novel role for WIP1 in the control of basal glucose homeostasis and response to a HFD challenge.

Acknowledgments

We thank Larry Donehower for the WIP1^KO animals and protocol for wip1 RT-PCR; Roger Davis of the Howard Hughes Medical Institute and University of Massachusetts Medical School, Worcester for critical reading of the manuscript; David Garlick of the University of Massachusetts Medical School, Worcester for histology analysis; and Julie Cavanagh of the University of Massachusetts Medical School, Worcester for assistance with real-time PCR. H.K.S. and J.K.K. are members of the UMass DERC (DK32520).

This work was supported by a grant from the Dean's Fund (H.K.S.), the National Institutes of Health Grant DK80756 (to J.K.K.) and the UMass Mouse Metabolic Phenotyping Center Grant U24-DK9300. Core resources supported by the Diabetes Endocrinology Research Center Grant DK32520 were also used.

Disclosure Summary: The authors have nothing to disclose.

Abbreviations

 
• AKT

  Ak mouse transforming

 
• ApoE

  apolipoprotein E

 
• GTT

  glucose tolerance test

 
• HFD

  high-fat diet

 
• HGP

  hepatic glucose production

 
• ITT

  insulin tolerance test

 
• LFD

  low-fat chow diet

 
• MEF

  murine embryo fibroblast

 
• p53

  tumor suppressor p53

 
• WAT

  white adipose tissue

 
• WIP1

  wild-type p53-inducible phosphatase

 
• WIP1^KO

  WIP1 knockout

 
• WT

  wild-type.