Myeloperoxidase Deletion Prevents High-Fat Diet–Induced Obesity and Insulin Resistance

Obesity, the most common cause of insulin resistance, is characterized by chronic, low-grade inflammation in insulin target tissues, including the liver, skeletal muscle, and adipose tissue (1,2). This is consistent with observations that the adipose tissue of obese mice and humans is infiltrated with many immune cells, including macrophages, T cells, B cells, and eosinophils (3–5). Adipose tissue macrophages secrete a variety of cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), that directly impair insulin signaling and lead to low insulin sensitivity. Recruitment of macrophages into adipose tissue is the initial event in obesity-induced inflammation and insulin resistance. In the early stage of obesity, neutrophils are recruited to adipose tissue, where they produce chemokines and cytokines, thereby promoting macrophage infiltration (6,7). Neutrophils can also promote an inflammatory response by releasing neutrophil elastase, a proteolytic enzyme produced during inflammation. Recently, several studies demonstrated that deletion of neutrophil elastase prevents high-fat diet (HFD)-induced insulin resistance and inhibits adipose tissue inflammation, with a reduction in adipose tissue neutrophils and macrophages (8,9), suggesting that neutrophils may participate in inflammation-induced metabolic disorders. In addition to neutrophil elastase, myeloperoxidase (MPO) is abundantly expressed in neutrophils and has been implicated in the initiation of the inflammatory response in adipose tissue. We therefore hypothesized that MPO might participate in obesity-induced insulin resistance.

MPO is a chlorinating oxidant-generating enzyme that initiates an acute inflammatory response and propagates chronic inflammation through the generation of pro-oxidants, including hypochlorous acid (HOCl) and tyrosyl radicals (10,11). Bacterial invasion or pathological stress induces MPO-catalyzed synthesis of HOCl from H₂O₂ and Cl ions in neutrophils. MPO-derived HOCl directly modifies lipoproteins and enhances their affinity for macrophages and the vessel wall, facilitating the development of vascular inflammation (12). A recent animal study demonstrated that neutrophils infiltrate adipose tissue in the early stage of HFD administration and release various substances, including reactive oxygen species, TNF-α, and MPO, all of which have the capacity to induce inflammation (13). Observations in humans also show that neutrophil counts in peripheral blood are increased in obese individuals and in type 2 diabetic patients, and importantly, these patients have higher plasma levels of MPO (14), suggesting a positive correlation between activation of MPO and metabolic disorders (15,16). Thus, MPO was considered an early biomarker of inflammation and cardiovascular risk factor in obese individuals; however, the functional roles of elevated MPO expression in adipose tissue in the pathogenesis of insulin resistance remain to be determined.

In the current study, we sought to determine whether MPO activation is involved in the development of obesity-associated insulin resistance. We found that consumption of an HFD induced neutrophil infiltration and MPO activation in adipose tissue and that knockout of MPO prevented HFD-induced obesity and insulin resistance.

HOCl and 4-aminobenzoic acid hydrazide (ABAH) were purchased from Sigma-Aldrich (St. Louis, MO). Protein A/G-agarose, radioimmunoprecipitation assay (RIPA) lysis buffer, and antibodies against β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-IR-β (Tyr1150/1151), IR-β, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, and horseradish peroxidase–linked secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibody against MPO was from R&D Systems (Minneapolis, MN). Antibodies against nitrotyrosine (NT), uncoupling proteins 1 (UCP1), and UCP3 were from Millipore Corp. (Billerica, MA). The enhanced chemiluminescence detection kit was obtained from Pierce (Rockford, IL).

MPO knockout (MPO^−/−) mice from The Jackson Laboratory (Bar Harbor, ME) were originally generated on the 129/SvJ background and were backcrossed onto C57BL/6J background for more than 10 generations (17). C57BL/6J mice were used as the wild-type (WT) control. Mice were housed in temperature-controlled cages under a 12-h light/12-h dark cycle. Starting at 6 weeks of age, male mice were fed an HFD (D12492; Research Diets, New Brunswick, NJ) consisting of 60% fat, 20% protein, and 20% carbohydrate, or a normal chow diet (ND) consisting of 13% fat, 29% protein, and 58% carbohydrates (LabDiet, St. Louis, MO) for up to 16 weeks. Body weight and blood glucose were monitored every 2 weeks. At the end of the experiments, mice were fasted for 6 h. Plasma, epididymal white adipose tissue (WAT), brown adipose tissue (BAT), and the liver were collected and stored at −80°C. The animal protocol was reviewed and approved by the University of Oklahoma Institutional Animal Care and Use Committee.

Mice were fasted overnight, and a glucose tolerance test (GTT) was performed by intraperitoneal injection of 1 g/kg body weight of 10% d-glucose, followed by measurement of blood glucose levels at 0, 30, 60, 90, and 120 min with a glucometer. To assess insulin tolerance, mice were fasted for 6 h and intraperitoneally injected with 0.75 units/kg insulin (Humulin R; Eli Lilly and Company, Indianapolis, IN). Blood glucose was measured at 0, 15, 30, 60, 90, and 120 min after injection. Plasma insulin concentrations were measured using an ELISA kit (ALPCO, Salem, NH). Plasma concentrations of triglyceride and cholesterol were measured using an Infinity kit (Thermo Fisher Scientific, Waltham, MA).

WT and MPO^−/− mice were intraperitoneally injected with 4% thioglycollate (1.5 mL) to induce chemical peritonitis. The lavage was collected 6 hours after the injection, and neutrophils were isolated by Ficoll gradient centrifugation (13). Monocytes were collected 12 h after the injection and purified by adherence to culture dish in DMEM medium containing 10% FBS. Peritoneal macrophages were collected 3 days after the injection and purified by adherence to culture dish in DMEM medium containing 10% FBS. Nonadherent cells were removed by washing with PBS.

Peroxidase activity in neutrophils and adipose tissue was measured by the chemiluminescence assay using luminol plus near-infrared quantum dots, as previously described (18). Briefly, epididymal WAT and neutrophils were homogenized with RIPA buffer containing 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentration was determined using the bicinchoninic acid method. The protein concentrations of homogenates were adjusted to 2 mg/mL and 1 mg/mL, respectively. Eighty microliters protein lysate were placed in a Costar 96-well black plate with 80 µL 2.3 mmol/L luminol (Thermo Fisher, Rockford, IL) and 1 µL 8 μmol/L QD800 (Invitrogen, Grand Island, NY). Then, 80 μL 2 mmol/L H₂O₂ were added to the mixtures to trigger production of HOCl. Luminescence was recorded for 20 seconds after the H₂O₂ addition to estimate peroxidase activity by using an M1000 microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

Mitochondria were isolated from BAT by differential centrifugation as described (19). The mitochondrial fraction was suspended in buffer consisting of 5 mmol/L MgCl₂, 215 mmol/L d-mannitol, 6.25 mmol/L KH₂PO₄, 20 μmol/L EGTA, 75 mmol/L sucrose, 20 mmol/L HEPES, and 0.1% BSA (pH 7.4). Forty microliters of 3 mg/mL mitochondrial protein were placed in a sealed chamber for measurement of oxygen consumption using a Clark-type oxygen electrode at 37°C (782 oxygen meter; Strathkelvin Instruments, North Lanarkshire, Scotland). Mitochondrial activation was initiated by addition of 0.25 mmol/L succinate and 1 mmol/L ADP. Oxygen consumption was monitored for 5 min before addition of 1 mmol/L KCN to stop the reaction.

To determine the UCP1-dependent oxygen consumption rate, mitochondrial respiration was initiated by addition of the substrates, including 30 μmol/L palmitoyl CoA or 5 mmol/L pyruvate, for 2 min, and then inhibited by adding 2 mmol/L guanosine 5′ diphosphate (GDP) for 2 min. UCP1-dependent respiration was calculated as the differences between substrate-stimulated and GDP-inhibited oxygen consumption rates. Maximal oxygen consumption rates were calculated by adding 1.5 μmol/L FCCP (carbonyl cyanide 4-[trifluoromethoxy]phenylhydrazone); finally, the reaction was stopped by adding 5 µg/mL antimycin A plus 2 μmol/L rotenone.

BAT mitochondria were incubated with 0.25 mmol/L succinate and 1 mmol/L ADP, as described previously. After the incubation, reaction mixture was centrifuged at 10,000g for 5 min. ATP in the supernatant was assayed by Jasco high-performance liquid chromatography, as described previously (20).

Hematoxylin and eosin staining of WAT, BAT, and liver sections, and immunohistochemical staining using antibodies against CD68 and neutrophil elastase (Abcam) were performed as described previously (21).

Mouse 3T3-L1 preadipocytes from the American Type Culture Collection (Manassas, VA) were cultured and differentiated into adipocytes using 3T3-L1 adipocyte differentiation medium DMEM/F12 50:50 (Zen-Bio, Inc., Durham, NC) supplemented with isobutylmethylxanthine (0.5 mmol/L), dexamethasone (1 μmol/L), and insulin (1.5 µg/mL; pH 7.4) (22). Adipocytes were used 8–12 days after differentiation, when 90–95% of the cells exhibited adipocyte phenotype. After overnight incubation in DMEM (pH 7.4) supplemented with 0.1% BSA, penicillin (100 units/mL), and streptomycin (100 µg/mL), the 3T3-L1 adipocytes were treated with HOCl in culture medium (DMEM, pH 7.4) for 1 h at 37°C. The concentrations of HOCl were determined at 292 nm in 0.1 mol/L NaOH (ε = 350 [mol/L]^-1 ⋅ cm⁻¹) before use.

Proteins were extracted from 3T3-L1 cells or mouse tissues with RIPA buffer containing 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride, as described previously (21). Protein concentration was determined using the bicinchoninic acid method. A total of 500 μg protein was incubated with NT antibody overnight at 4°C. Immunoprecipitates were washed four times with lysis buffer, boiled in Laemmli buffer (Boston BioProducts, Inc., Ashland, MA) for 5 min, and analyzed by Western blotting. For the Western blot analysis, tissue or cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp.). Membranes were probed with specific antibodies and subsequently incubated with horseradish peroxidase–linked secondary antibodies. Proteins were visualized by using an enhanced chemiluminescence detection system.

Total RNA was isolated from BAT and WAT using the Qiagen RNeasy Mini Kit (Germantown, MD). PCR primers were designed to specifically span an intron of target genes to ensure only cDNA but not genomic DNA was amplified. The primer sequences are detailed in Supplementary Table 1. To validate the primers, we ran the PCR products on agarose gel, which revealed single bands of the correct size. To determine the efficiency of the primers, we ran standard curves. The slopes of the standard curves (Ct vs. concentration) were close to −3.32. For reverse transcription, 1 µg total mRNA was converted to first-strand cDNA in 20-µL reactions using a cDNA synthesis Kit (Promega). Quantitative real-time PCR was performed using SYBR Green (Invitrogen). The relative RNA amount was calculated with the 2^−ΔΔCt method and normalized with 18S rRNA as the internal control.

To determine HOCl-induced generation of peroxynitrite (ONOO⁻) in 3T3-L1 adipocytes, the cells were cultured in DMEM (pH 7.4), treated with HOCl (200 μmol/L) for 1 h, and then incubated in the culture medium containing dihydrorhodamine (5 μmol/L). After incubation for 1 h, 200 μL medium was collected to a 96-well plate. The formation of rhodamine 123 was monitored by fluorescence spectroscopy, and the excitation and emission wavelengths were 500 and 570 nm, respectively.

Values are expressed as mean ± SEM. One-way or two-way ANOVA was used to compare the differences among three or more groups. P < 0.05 was considered statistically significant.

Obesity-associated tissue inflammation is a major cause of insulin resistance. Consistent with previous findings that adipose tissue of obese mice and humans is infiltrated with large numbers of immune cells, many neutrophils with pale pink cytoplasm and lobed nuclei were observed in epididymal WAT from HFD-fed mice, whereas infiltration of neutrophils was rarely found in adipose tissue from ND-fed mice (Fig. 1A). Because MPO is a major enzyme in neutrophils, we examined whether HFD feeding regulates peroxidase activity. MPO expression was upregulated in epididymal WAT from mice after 16 weeks of HFD feeding (Fig. 1B). The increase in MPO protein levels was associated with an increase in peroxidase activity, as determined by luminescent excitation of near-infrared nanoparticles (Fig. 1C) (18).

MPO is not expressed in adipocytes; therefore, the increases in MPO protein levels and high peroxidase activity most likely results from infiltrated immune cells. To identify which immune cells contributed to the HFD-enhanced peroxidase activity, we first isolated neutrophils, monocytes, and macrophages from peritoneal lavage after thioglycollate administration and measured peroxidase activity in these cells. Peroxidase activity in neutrophils was 15-fold higher than that in peritoneal cells isolated from the mice treated with normal saline. Meanwhile, peroxidase activity was increased by 3.5-fold in monocytes and by 1.7-fold in macrophages (Fig. 1D).

Next, we analyzed MPO expression in neutrophils and macrophages isolated from the stromal vascular fraction of epididymal WAT in HFD-fed WT mice by using flow cytometry. FACS revealed that the percentages of neutrophils (Ly6G⁺CD11b⁺ cells) and macrophages (F4/80⁺CD11b⁺ cells) in HFD-fed mice were higher than those in ND-fed mice (neutrophils: 0.72 ± 0.27% vs. 0.07 ± 0.005%; macrophages: 10.1 ± 0.6% vs. 6.7 ± 1.5%, n = 6; P < 0.05). The MPO level was significantly higher in neutrophils than in macrophages (Fig. 1E). We further examined whether HFD feeding enhanced peroxidase activity in neutrophils. As expected, the peroxidase activity of neutrophils in HFD-fed mice was twofold higher than that in ND-fed mice (Fig. 1F). These data suggest that infiltration of neutrophils in WAT is the major source of HFD-enhanced peroxidase activity and that activation of MPO may be a mechanism underlying HFD-induced obesity and insulin resistance.

In addition to increasing infiltration of neutrophils in WAT, consumption of the HFD also increased neutrophil infiltration in the liver (Fig. 1G and H) and BAT (Fig. 1I and J). Similarly, the HFD also increased infiltration of macrophages in the liver (Fig. 1K and L) and BAT (Fig. 1M and N).

Next, we examined whether deletion of MPO would affect basic metabolic function using MPO^−/− mice. Deletion of the MPO gene abolished MPO protein expression (Fig. 2A) and peroxidase activity (Fig. 2B) in neutrophils. Although HFD feeding did not enhance MPO protein levels in WT mouse neutrophils (Fig. 2A), it significantly increased peroxidase activity. This increase was absent in HFD-fed MPO^−/− mouse neutrophils (Fig. 2B). Metabolic cages were used to monitor the metabolic changes after HFD feeding. WT and MPO^−/− mice had similar energy and water intake (Supplementary Fig. 1A and B). Although there was no difference in body weight gain between ND-fed WT and MPO^−/− mice, deletion of MPO significantly attenuated HFD-enhanced body weight (Fig. 2C). The body weight gains of WT and MPO^−/− mice were similar in the early stage of HFD feeding; however, the WT mice gained more body weight than MPO^−/− mice after 10 weeks of HFD feeding. Accordingly, HFD-fed WT mice gained more epididymal WAT, but not perirenal fat, than HFD-fed MPO^−/− mice (Fig. 2D). Thus, MPO deficiency was resistant to HFD-induced obesity even though it did not affect energy intake.

Because deletion of MPO attenuated HFD-enhanced body weight without affecting energy intake, we examined whether deletion of MPO resulted in less body weight gain in HFD-fed mice through increasing energy expenditure in BAT, a major tissue in which energy is dissipated to maintain body temperature. In agreement with the findings that neutrophil-specific elastase knockout mice have a higher core temperature and oxygen consumption (23), the rectal temperature of HFD-fed MPO^−/− mice was higher than that of WT mice (Fig. 2E).

To determine the mechanism underlying the higher temperature in HFD-fed MPO^−/− mice, we examined the mRNA and protein levels of UCP1, UCP2, and UCP3, three major uncoupling proteins in BAT, which dissipate oxidative energy as heat in BAT mitochondria (24). UCP1 and UCP3, but not UCP2 (data not shown), mRNA levels were significantly higher in HFD-fed MPO^−/− mice than in HFD-fed WT mice (Fig. 2F and G). At the protein level, UCP1 expression was higher in MPO^−/− mice than in WT mice; however, UCP3 protein levels were not significantly different in WT and MPO^−/− mice (Fig. 2H and I). In BAT, UCP1 acts as a proton carrier that dissipates the proton gradient generated in oxidative phosphorylation. Activation of UCP1 enhances mitochondrial respiration and uncouples the respiratory chain from ATP synthase, leading to the dissipation of oxidation energy as heat (25). We, therefore, measured oxygen consumption in mitochondria isolated from HFD-fed WT and MPO^−/− mouse BAT using succinate as a substrate. The mitochondrial oxygen consumption of MPO^−/− mice was higher than that of WT mice (Fig. 2J); however, mitochondria produced similar amounts of ATP in WT and MPO^−/− BAT (Fig. 2K). UCP1 in BAT was activated by free fatty acids (e.g., palmitoyl-CoA) and carbohydrate (e.g., pyruvate) but inhibited by GDP (26,27).

We further analyzed the oxygen consumption rate after addition of palmitoyl-CoA, pyruvate, and GDP. Addition of palmitoyl-CoA or pyruvate significantly increased oxygen consumption rate in MPO^−/− mice compared with WT mice (Fig. 2L and M). The increase in the oxygen consumption rate was inhibited by addition of GDP, which inhibits UCP1 activity (Fig. 2L and M). In addition, the UCP1-dependent oxygen consumption rate, estimated by the GDP-inhibitable oxygen consumption rate, was higher in MPO^−/− mice than in WT mice after addition of palmitoyl-CoA or pyruvate (Fig. 2N). These results suggest that upregulation of UCP1 promotes mitochondrial oxygen consumption and increases body temperature in MPO^−/− mice.

MPO has been reported to initiate acute inflammation and propagate chronic inflammation in multiple organs. We therefore investigated whether deletion of MPO alleviates HFD-induced inflammation in WAT. As demonstrated by immunohistochemical staining of the macrophage marker CD68, the extent of macrophage infiltration in epididymal WAT was greater in HFD-fed WT than in HFD-fed MPO^−/− mice. A characteristic crown-like structure that contains neutrophils and macrophages could often be observed in HFD-fed mice but was rarely observed in HFD-fed MPO^−/− mice (Fig. 3A and B). Consistently, high-fat feeding dramatically increased the expression of F4/80 mRNA, another macrophage marker, in WT epididymal WAT, and the increase was significantly diminished in HFD-fed MPO^−/− WAT (Fig. 3C).

We further analyzed the mRNA levels of proinflammatory cytokines, including TNF-α, IL-6 and IL-1β, MCP-1, chemokine (C-X-C motif) ligand 14 (CXCL14), and chemokine receptor type 2 (CCR2), in WAT of HFD-fed WT and MPO^−/− mice, and found that the deletion of MPO reduced HFD-enhanced proinflammatory cytokine expression (Fig. 3D). After 6 weeks of high-fat feeding, the expression of inducible nitric oxide synthase (iNOS) mRNA in MPO^−/− mice tended to be lower than that in WT mice, although there was no statistical significance between these mice. We further analyzed protein expression of iNOS in WAT after 16 weeks of high-fat feeding. Immnohistochemical staining demonstrated that the expression of iNOS protein was significantly lower in MPO^−/− mice than that in WT mice (Fig. 3E).

Obesity is a major risk factor for insulin resistance. Because MPO^−/− mice were resistant to HFD-enhanced obesity, we investigated whether deletion of MPO affects insulin sensitivity. In WT mice, 16 weeks of HFD feeding resulted in an increase in blood glucose and a reduction in plasma insulin levels. Conversely, MPO^−/− mice showed significantly lower levels of blood glucose compared with WT mice (Fig. 4A). In addition, after 6 weeks of HFD feeding, MPO^−/− mice had lower levels of plasma insulin and fasting blood glucose than did WT mice (Fig. 4B and C); however, plasma concentrations of triglycerides and cholesterol were not significantly different in the two groups (Fig. 4D and E). We further analyzed insulin sensitivity by performing GTTs and insulin tolerance tests in WT and MPO^−/− mice after 6 or 16 weeks of HFD feeding. The HFD-fed MPO^−/− mice showed greater tolerance to the glucose challenge (Fig. 4F, G, I, and J) and better sensitivity to insulin stimulation (Fig. 4H and K). These results suggest that MPO deficiency attenuates HFD-impaired insulin sensitivity.

To determine whether MPO affects insulin signaling in epididymal WAT of HFD-fed mice, we analyzed insulin-stimulated phosphorylation of insulin receptor-β (IR-β) and Akt in the animals. As shown in Fig. 5A–D, insulin stimulation increased phosphorylation of Akt at Thr308 and Ser473 in WAT isolated from ND-fed WT and MPO^−/− mice. HFD feeding inhibited insulin-stimulated phosphorylation of Akt in WT mice. However, the inhibitory effect of the HFD on Akt phosphorylation was absent in MPO^−/− mice, confirming that deletion of MPO improves insulin sensitivity in HFD-fed mice. Similarly, insulin-stimulated phosphorylation of IR-β was inhibited in HFD-fed WT mice, and inhibition of IR-β phosphorylation was absent in HFD-fed MPO^−/− mice (Fig. 5C and D). Thus, MPO deletion prevented HFD-impaired insulin signaling.

We also analyzed the effect of the HFD on insulin signaling in the liver. Similar to the observations in epididymal WAT, insulin stimulation increased phosphorylation of Akt at Thr308 and Ser473 in the livers of ND-fed WT and MPO^−/− mice. The increase in Akt phosphorylation was attenuated in HFD-fed WT mice but not in MPO^−/− mice (Fig. 5E). Insulin-stimulated phosphorylation of IR-β was also inhibited in HFD-fed WT mice, and the inhibition was absent in HFD-fed MPO^−/− mice (Fig. 5F).

We further investigated whether pharmacological inhibition of MPO prevents HFD-induced insulin resistance. Chronic administration of the MPO inhibitor ABAH dose-dependently inhibited peroxidase activity of neutrophils in WT mice (Fig. 6A), with 50% inhibition at 40 mg/kg. This dose was then intraperitoneally injected into HFD-fed WT mice. After 1 or 2 weeks of treatment, GTT and insulin tolerance test analysis was done. The results showed that ABAH treatment improved insulin sensitivity and enhanced glucose tolerance in insulin-resistant, obese WT mice compared with DMSO-treated mice (Fig. 6B–E). Moreover, ABAH did not improve glucose tolerance and insulin sensitivity in MPO^−/− mice (Fig. 6F and G).

Next, we determined whether deletion of MPO prevents HFD-reduced phosphorylation of IR-β through regulation of IR-β expression. As shown in Fig. 7A, IR-β levels were decreased in epididymal WAT from HFD-fed WT mice compared with ND-fed controls. Meanwhile, deletion of MPO attenuated the reduction in IR-β protein levels associated with HFD. However, IR-β mRNA expression was not affected by HFD feeding in WT and MPO^−/− mice (Fig. 7B). Thus, HFD may regulate IR-β expression through posttranscriptional modification of the protein (28). MPO was reported to oxidize and nitrate proteins at tyrosine. Tyrosine nitration of a protein may alter the protein structure and accelerate its degradation (29). Thus, we hypothesized that activation of MPO induces tyrosine nitration of IR-β and subsequently accelerates its degradation. To test this hypothesis, we first detected 3-NT (3-NT) formation in epididymal WAT by using immunohistochemistry. High-fat feeding significantly increased 3-NT formation in WT mice, but this increase was attenuated in MPO^−/− mice (Fig. 7C and D). Next, 3-NT was immunoprecipitated from homogenates of epididymal WAT, and IR-β was detected by Western blotting. The level of tyrosine nitration of IR-β was higher in HFD-fed WT mice than in HFD-fed MPO^−/− mice (Fig. 7E). To determine whether MPO-produced HOCl mediates tyrosine nitration of IR-β in HFD-fed mice, tyrosine nitration of IR-β was detected in ABAH-treated and HFD-fed mice. Inhibition of HOCl production by administration of ABAH reduced tyrosine nitration of IR-β in HFD-fed mice (Fig. 7F).

HOCl is a major chlorinating oxidant product of MPO; thus, we investigated whether MPO-derived HOCl contributes to insulin resistance in vitro. In 3T3-L1 adipocytes, HOCl pretreatment inhibited insulin-stimulated phosphorylation of IR-β, insulin receptor substrate-1 (IRS-1), and Akt, indicating impairment of insulin signaling (Fig. 7G). HOCl also inhibited insulin-induced binding of IRS-1 to P85, the regulatory subunit of phosphatidylinositide 3-kinase, which is the key enzyme to activate phosphoinositide-dependent kinase-1 and Akt during insulin signaling (Fig. 7H). HOCl has been reported to induce the production of ONOO⁻, a potent oxidant that nitrates proteins at tyrosine residues in endothelial cells (30). We therefore investigate if endogenous ONOO⁻ production was involved in HOCl-induced tyrosine nitration of IR-β in 3T3-L1 adipocytes. We found that HOCl treatment dramatically increased IR-β tyrosine nitration in the presence or absence of insulin. In contrast, HOCl treatment had little effect on tyrosine nitration of IRS-1 and Akt (data not shown). Administration of superoxide dismutase 1 (SOD1) to remove superoxide, L-NAME to inhibit nitric oxide production, and uric acid to scavenge ONOO⁻, all prevented HOCl-induced IR-β tyrosine nitration (Fig. 7I) although they had no effects on the binding between IRS-1 and P85 (Fig. 7H). We further examined the effect of HOCl on ONOO⁻ production in 3T3-L1 adipocytes. Exposure of cells to HOCl significantly enhanced ONOO⁻ production in the presence and absence of insulin. The overproduction of ONOO⁻ was abolished by overexpression of SOD1 or administration of L-NAME or uric acid. However, overexpression of catalase failed to prevent HOCl-induced overproduction of ONOO⁻ (Fig. 7J). Taken together, our data suggest that ONOO⁻ mediates HOCI-induced tyrosine nitration of IR-β.

Obesity is characterized by immune cell infiltration in adipose tissue and high levels of proinflammatory molecules in the circulation. These proinflammatory mediators may impair insulin signaling, resulting in insulin resistance. MPO is an enzyme released from neutrophils during inflammation and has been implicated in the development of obesity and insulin resistance (15). However, the molecular mechanisms by which MPO promotes the development of obesity and insulin resistance have not been established. The current study demonstrates that obese mice have high MPO protein levels and activity in adipose tissues and neutrophils. Genetic deletion of MPO led to less body weight gain, inhibition of inflammation, and improvement of insulin sensitivity and glucose tolerance in HFD-fed mice. The improvement of insulin sensitivity is associated with the restoration of IR-β protein levels in HFD-fed mice (Fig. 7K).

Emerging evidence suggests that activation of neutrophils is involved in the development of obesity and obesity-associated insulin resistance. In obese and type 2 diabetic patients, circulating neutrophil counts are significantly increased (31) in association with increased oxidative stress and inflammation (7). In HFD-fed mice, adipose tissue was infiltrated with neutrophils after 3 days of HFD feeding (13,32), and neutrophil infiltration was maintained throughout the 90-day study period (9). Suppression of HFD-induced neutrophil infiltration by deletion of neutrophil elastase led to improved glucose tolerance and increased insulin sensitivity. Consistent with these findings, we observed neutrophil accumulation in adipose tissue of HFD-fed mice, which was accompanied by enhanced body weight gain and impaired insulin sensitivity. Many inflammatory stimuli can stimulate neutrophils to release the contents of their granules, including MPO, neutrophil elastase, and proteinases, into the surrounding tissues to induce acute inflammation. Our data indicate that the infiltrating polymorphonuclear neutrophils are the major source of MPO activity in adipose tissue of HFD-fed mice and that deletion of MPO attenuated the HFD-enhanced inflammatory response and inhibited HFD-induced insulin resistance. Thus, all evidence supports the view that neutrophil infiltration of adipose tissue contributes to insulin resistance in obesity (33).

MPO may promote tyrosine nitration of proteins, leading to alteration of protein structure and function. Nitration of a tyrosine residue either prevents subsequent phosphorylation of the residue or stimulates its phosphorylation, resulting in constitutively active protein. In addition, tyrosine nitration may alter the rate of proteolytic degradation of nitrated proteins, leading to faster degradation or accumulation of the nitrated proteins in cells. Tyrosine nitration of proteins by MPO has been reported in lung (34), blood, and vasculature (35). MPO chlorinates and nitrates Tyr192 of apolipoprotein A-I, leading to impaired cholesterol efflux (35,36). In vascular endothelial cells, MPO and HOCl increase ONOO⁻ production (37), resulting in endothelial NOS uncoupling and exacerbating oxidative stress (30). Moreover, tyrosine nitration of protein has been shown to be associated with a strong inflammatory response in human atherosclerotic plaques (38) and retina (39). In agreement with the previous findings that tyrosine nitration of the insulin-signaling molecules, including IRS-1 and Akt, contributes to HFD-induced insulin resistance, we found that HFD feeding resulted in tyrosine nitration of IR-β, which was associated with a reduction in IR-β protein and phosphorylation levels and also with impaired insulin signaling. Deletion of MPO prevented HFD-induced tyrosine nitration of IR-β, restored IR-β protein expression, and improved insulin sensitivity. Whether tyrosine nitration of IR-β accelerates its degradation warrants further investigation.

Consistent with the findings that high-fat feeding results in less body weight gain and higher body temperature and oxygen consumption in neutrophil elastase knockout mice than in control mice (8), we observed less body weight gain in MPO^−/− mice after HFD feeding, with no effect on energy intake. Deletion of MPO also led to higher levels of UCP1 expression in BAT. UCP1 is a proton transporter of the mitochondrial inner membrane that uncouples oxidative metabolism from ATP synthesis and dissipates energy as heat. UCP1 has been reported to play important roles in energy homeostasis, and ablation of UCP1 prevented Western diet–induced obesity (40). In agreement with the upregulation of UCP1, rectal temperature and oxygen consumption measured in isolated BAT mitochondria were increased in the current study, but ATP production did not increase in HFD-fed MPO^−/− mice, suggesting the uncoupling of oxidative metabolism and ATP synthesis and increase in thermogenesis, which may account for the smaller body weight gain in HFD-fed MPO^−/− mice. Further investigations are warranted to elucidate the mechanism by which MPO regulates UCP1 expression and consequent thermogenesis and energy metabolism. Because loss of body weight and fat mass almost always improves insulin sensitivity (41), which inhibits the Jun NH₂-terminal kinase and inhibitor of κB kinase–nuclear factor-κB pathways and reduces inflammation (42), the smaller body weight gain in HFD-fed MPO^−/− mice may also contribute to the improvement of insulin resistance in these mice.

In summary, activation of MPO was a critical event in HFD-induced obesity and insulin resistance. Deletion of MPO reduced body weight gain through the upregulation of UCP1 in BAT. In addition, MPO deficiency is associated with a restoration of IR-β protein levels and improved insulin sensitivity in HFD-fed mice. Thus, inhibition of MPO may be a potential strategy for prevention and treatment of obesity and its complications.

Funding. This work was supported by National Institutes of Health grants (HL-079584, HL-080499, HL-074399, HL-089920, HL-096032, HL-10488, AG047776, and HL-105157), a research award from the American Diabetes Association, and funds from Warren Chair in Diabetes Research from the University of Oklahoma Health Sciences Center. Q.W. is a recipient of an American Heart Association Postdoctoral fellowship. M.-H.Z. is a recipient of the National Established Investigator Award of the American Heart Association.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Q.W. contributed to the study design, performed experiments, and wrote the manuscript. Z.X. designed experiments and wrote the manuscript. W.Z., J.Z., Y.W., M.Z., and H.Z. performed some experiments. M.-H.Z. contributed to the study design and interpretation and wrote the manuscript. M.-H.Z. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.