Peroxiredoxin 6, a novel player in the pathogenesis of diabetes.

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Diabetes Volume 63, October 2014

Francesca Pacifici,1 Roberto Arriga,1 Gian Pio Sorice,2,3 Barbara Capuani,1 Maria Giovanna Scioli,4 Donatella Pastore,1 Giulia Donadel,1 Alfonso Bellia,1 Sara Caratelli,1,5 Andrea Coppola,1 Francesca Ferrelli,1 Massimo Federici,1 Giuseppe Sconocchia,1,5 Manfredi Tesauro,1 Paolo Sbraccia,1 David Della-Morte,1,6 Andrea Giaccari,2,7 Augusto Orlandi,4 and Davide Lauro1

Peroxiredoxin 6, a Novel Player in the Pathogenesis of Diabetes

METABOLISM

Diabetes 2014;63:3210–3220 | DOI: 10.2337/db14-0144

Enhanced oxidative stress contributes to the pathogenesis of diabetes and its complications. Peroxiredoxin 6 (PRDX6) is a key regulator of cellular redox balance, with the peculiar ability to neutralize peroxides, peroxynitrite, and phospholipid hydroperoxides. In the current study, we aimed to define the role of PRDX6 in the pathophysiology of type 2 diabetes (T2D) using PRDX6 knockout (2/2) mice. Glucose and insulin responses were evaluated respectively by intraperitoneal glucose and insulin tolerance tests. Peripheral insulin sensitivity was analyzed by euglycemic-hyperinsulinemic clamp, and molecular tools were used to investigate insulin signaling. Moreover, inflammatory and lipid parameters were evaluated. We demonstrated that PRDX62/2 mice developed a phenotype similar to early-stage T2D caused by both reduced glucose-dependent insulin secretion and increased insulin resistance. Impaired insulin signaling was present in PRDX62/2 mice, leading to reduction of muscle glucose uptake. Morphological and ultrastructural changes were observed in islets of Langerhans and livers of mutant animals, as well as altered plasma lipid profiles and inflammatory parameters. In conclusion, we demonstrated that PRDX6 is a key mediator of overt hyperglycemia in T2D glucose metabolism, opening new perspectives for targeted therapeutic strategies in diabetes care.

by an imbalance between reactive oxygen species (ROS) production and antioxidant defense systems. Among all body tissues, pancreatic b-cells are very sensitive to oxidative stress because of their low expression of antioxidant enzymes like superoxide dismutase (SOD) and glutathione peroxidase (2). Moreover, hyperglycemia by itself induces IR, increasing oxidative stress injuries, which lead to overt type 2 diabetes (T2D) (3). Interestingly, a relatively new family of antioxidant proteins, the peroxiredoxins (PRDXs), is more highly expressed in pancreatic b-cells (4). Among the six members of this non-seleno peroxidase family, PRDX6 is present in the cytoplasm and is unique because it has peroxidase and also phospholipase A2 activity (5). Several findings demonstrate the importance of PRDX6 in maintaining redox homeostasis, as follows: lack of PRDX6, in fact, increases the susceptibility to oxidative stress in different tissues (6,7). Nevertheless, data on the relationship between PRDX6 and the pathogenesis of IR and T2D are not available (8). Therefore, we hypothesized that, in terms of physiological status, PRDX6 may play a role in the etiology of IR and diabetes conditions through tissue redox levels. In the current study, we tested our hypothesis in a model of PRDX6 knockout mice (PRDX62/2). RESEARCH DESIGN AND METHODS Animal Models

A large body of evidence supports a pivotal role for oxidative stress in the etiopathogenesis of insulin resistance (IR) and diabetes (1). Oxidative stress is characterized

C57BL/6J wild-type (WT) mice weighing 18–20 g were obtained from The Jackson Laboratory (Bar Harbor, ME), while PRDX62/2 mice of mixed background (C57BL6/129SvJ) were provided by Professor Xiaosong Wang (The Jackson

1Department

Corresponding author: Davide Lauro, [email protected].

of System Medicine, University of Rome Tor Vergata, Rome, Italy of Endocrinology and Metabolic Diseases, Università Cattolica del Sacro Cuore, Rome, Italy 3Diabetic Care Clinics, Associazione dei Cavalieri Italiani Sovrano Militare Ordine di Malta, Rome, Italy 4Anatomic Pathology, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 5Institute of Translational Pharmacology, National Research Council, Rome, Italy 6Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Pisana, Rome, Italy 7Fondazione Don Carlo Gnocchi, Milan, Italy 2Division

Received 27 January 2014 and accepted 28 April 2014. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0144/-/DC1. F.P. and R.A. contributed equally to this work. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

diabetes.diabetesjournals.org

Laboratory) (6). All mice were housed in a temperaturecontrolled animal room with a 12-h light-dark cycle, and were given free access to commercial mouse chow and water. Three-month-old male mice were used for all experiments. Five animals were used in each experimental group. Glucose and Insulin Tolerance Tests and Insulin Levels

An intraperitoneal glucose tolerance test (IPGTT) was performed with 2 g/kg glucose injection in mice fasted for 16 h. Glucose levels were measured after 0, 30, 60, 90, and 120 min using an automated OneTouch Glucometer (LifeScan, Milpitas, CA). Insulin concentration was quantified on plasma samples with a Mouse Insulin ELISA Kit (Mercodia, Uppsala, Sweden). An insulin tolerance test (ITT) was performed using intraperitoneal injection of 0.75 international units (IU)/kg insulin in mice fasted for 4 h, and glucose levels were measured after 0, 15, 30, and 60 min. Blood samples were obtained from the retro-orbital sinus. Animal studies were approved by the University of Rome Tor Vergata Animal Care and Use Committee. Euglycemic-Hyperinsulinemic Clamp Studies

Insulin clamp studies were performed as previously described (9). Briefly, 3 to 5 days prior to the clamp, a catheter was inserted into the right internal jugular vein and extended to the level of the right atrium. At time zero, after mice were fasted for 6 h, a primed continuous infusion of human insulin (18.0 mU/kg/min; Actrapid 100 IU; Novo Nordisk, Copenhagen, Denmark) was started simultaneously with a variable infusion of 20% dextrose in order to maintain the plasma glucose concentration constant at its basal level (80–100 mg/dL). Blood samples (;2 mL) were obtained from the tail vein at 10-min intervals for at least 2 h. Average glucose concentrations and glucose infusion rates were measured during the last 30 min of the steady-state clamp period.

Pacifici and Associates

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insulin (1 unit/kg) was injected through the portal vein. Five minutes after injection, mice were killed, and the gastrocnemius was excised and frozen immediately in liquid nitrogen. Subsequently, gastrocnemius samples were homogenized with 1 mL cold extraction buffer containing 20 mmol/L Tris (pH 7.6), 137 mmol/L NaCl, 1.5% NP40, 1 mmol/L MgCl2, 1 mmol/L CaCl2, glycerol 10%, 2 mmol/L phenylmethylsulfonyl fluoride, 13 protease inhibitor cocktail (Roche, Indianapolis, IN), 2 mmol/L Na3VO4, and 2 mmol/L EDTA using a dounce homogenizer. The homogenates were maintained on ice for 30 min and then were centrifuged at 14,000 rpm for 30 min. Supernatants were collected and stored at 280°C, while pellets, containing membrane proteins fractions, were resuspended in cold extraction buffer, maintained on ice for 30 min, and subsequently centrifuged at 50,000 rpm for 1 h. Supernatants, containing membrane proteins, were maintained at 280°C before analysis. Protein concentration was determined using a Bradford assay (Bio-Rad Laboratories, Hercules, CA), loaded on precast 4–15% gels (Bio-Rad Laboratories), separated by SDS-PAGE, and then transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (BioRad Laboratories). Afterward, membranes were incubated with antibodies directed against phospho-Akt-1 Ser473, phospho stress-activated protein kinase/Jun NH2-terminal kinase (JNK) (Cell Signaling Technology), phospho-Akt-2 Ser474, GLUT4 (Abcam), tubulin (Sigma-Aldrich), Akt-1, Akt-2, and stress-activated protein kinase/JNK (Cell Signaling Technology). The antigen-antibody complexes were detected with enhanced chemiluminescence (GE Healthcare), followed by exposure on blotting to X-ray film. Bands were quantified using Gel Doc XR+ with Image Lab Software (Bio-Rad Laboratories). Immunoprecipitation

Pancreatic islets extraction was performed according to Carter et al. (10). Mice were anesthetized with Avertin 5 mg/10 g body weight and killed. Pancreata were perfused with collagenase P after the common bile duct was clamped, and then were incubated for 8 min at 37°C in cold Hanks’ balanced salt solution. Digested pancreata were washed three times with G solution (Hanks’ balanced salt solution plus 1% BSA) and subsequently filtered. After centrifugation, pellets were resuspended in 10 mL Histopaque 1100 solution and centrifuged for 20 min at 1,200 rpm. Afterward, islets were washed three times and then were transferred into a sterile Petri dish containing 5 mL RPMI 1640 medium with 10% FBS and penicillin/streptomycin. Islets were cultured for 24 h to allow recovery from the stress associated with the isolation process.

Insulin receptor substrate (IRS) 1, IR b-subunit, and phosphoinositide 3-kinase (PI3K) were immunoprecipitated using standard protocols. Briefly, antibodies against IRS1 or IR b-subunit were rocked for 1 h at 4°C with protein A and protein G, respectively (GE Healthcare), and centrifuged at 15,000g for 10 min. Then, 1 mg muscle lysates were added to the complexes and were immunoprecipitated by rocking overnight at 4°C. Immunoprecipitates were washed three times, resuspended in 45 mL 23 LDS Sample Buffer (Invitrogen, Carlsbad, CA), and heated at 70°C for 10 min. Subsequently, immunoprecipitates were loaded on precast 4–12% gels (Invitrogen), separated by SDS-PAGE, and subjected to immunoblot analysis as reported above. Antibodies against phosphotyrosine RC20 (BD), PY20 (Santa Cruz Biotechnology), PI3K p85 a-subunit, insulin receptor b-chain (Cell Signaling Technology), and IRS1 (Millipore) were used.

Western Blot Analysis of Insulin Signaling and Subcellular Fractionation

Microscopic Evaluation of Nonalcoholic Steatohepatitis and Pancreatic Islets

Experiments were carried out in mice fasted overnight. Animals were anesthetized with Avertin (5 mg/10 g), and

Formalin-fixed paraffin 4-mm-thick hepatic and pancreatic tissue sections were cut and stained with hematoxylin-eosin

Pancreatic Islets Extraction

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PRDX6 and Pathogenesis of Diabetes

for microscopic examination (11). Liver microscopic features were grouped into five broad categories for the microscopic evaluation of nonalcoholic steatohepatitis (NASH), as already described (12). The number of pancreatic islets and the percentage of the islets area were calculated on images acquired by a digital camera (DXM1200F; Nikon Italia, Milan, Italy) and measured using Scion Image software (Scion Corporation, Frederick, MD) at a 320 magnification. All analyses were performed in a blinded fashion by two different pathologists, with an interobserver variability of ,5%. Gene Expression Analysis by Real-Time PCR

Total RNA from skeletal muscle, liver, and white adipose tissue (WAT) was isolated using TRIzol (Invitrogen). Two and one-half micrograms of total RNA was reverse transcribed into cDNA using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Qualitative real-time PCR was performed using an ABI PRISM 7500 System and TaqMan reagents (Applied Biosystems). Each reaction was performed in duplicate using standard conditions, and results were normalized with b-actin. The relative expression was calculated using the comparative DDCT method, and the values were expressed as 22DDCT (13). Statistical Analysis

Data were analyzed using Prism 5 (GraphPad, La Jolla, CA). All data are expressed as mean 6 SE. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post hoc test or unpaired one-tailed Student t test when appropriate. Values of P , 0.05 were considered statistically significant. RESULTS PRDX62/2 Mice Develop an Early Stage of Diabetes Linked With Higher Levels of IR

To evaluate the impact of PRDX6 deletion on glucose and insulin metabolism, PRDX62/2 and WT mice underwent an IPGTT and an ITT. As shown in Fig. 1A, after 30 min of glucose administration, and up to the end of the test, PRDX62/2 mice had significantly higher glucose values compared with WT mice (P , 0.05). Moreover, as demonstrated with the ITT, PRDX62/2 mice were insulin resistant, having significantly higher glucose values at 30 and 60 min after insulin injection compared with WT mice (P , 0.05) (Fig. 1B). These results were consistent with reduced peripheral insulin sensitivity, which is a condition of early-stage diabetes. In addition, during the IPGTT, plasma levels of insulin were significantly reduced after 15 min in PRDX62/2 mice compared with WT mice (P , 0.0005) (Fig. 1C), demonstrating an impaired insulin secretion. Although PRDX62/2 mice have lower insulin secretion after glucose load, insulin mRNA was significantly increased in pancreatic islets of PRDX62/2 mice compared with WT mice (Fig. 1D), suggesting higher synthesis of insulin in PRDX62/2 pancreatic b-cells. Interestingly, this defect of insulin secretion was also associated with a reduction


of pancreatic islet volume (Fig. 3). It is therefore possible that increased insulin production represents a compensatory mechanism for increasing insulin secretion by the residual pancreatic islets. Next, we sought to perform euglycemic-hyperinsulinemic clamp to quantify the amount of peripheral IR (Fig. 1E). During this procedure, the insulin concentration was raised to ;600 pmol/L, while the plasma glucose concentration was maintained at ;6 mmol/L through a variable infusion of glucose. We found a significant reduction of peripheral whole-body glucose disposal in PRDX62/2 mice compared with WT mice (P , 0.005). The decrease of the mean value in PRDX62/2 mice suggests that diminished insulin sensitivity could be located mainly at the skeletal muscle level, since ;80% of glucose uptake occurs in skeletal muscle during the test and PRDX6 is largely expressed in skeletal muscle (data not shown) (14). Insulin Signaling Is Impaired in Skeletal Muscle of PRDX62/2 Mice

To confirm this hypothesis, we analyzed phosphorylation and the activity of key components of the insulin cascade in skeletal muscle of PRDX62/2 and WT mice. After in vivo insulin stimulation, muscle insulin receptor was isolated with immunoprecipitation, and insulin-induced tyrosine phosphorylation was measured by immunoblotting with antiphosphotyrosine antibody. A significant increase in insulin receptor b-subunit phosphorylation was observed in PRDX62/2 mice compared with WT mice (P , 0.05) (Fig. 2A). Conversely, a significant reduction of IRS1 tyrosine phosphorylation after insulin stimulation was observed in PRDX62/2 mice (P , 0.005) (Fig. 2B). The IRS1-PI3K interaction was also evaluated, and a significant reduction in the amount of p85 was found in IRS1 immunoprecipitates from the muscles of PRDX62/2 mice (Fig. 2C). On the contrary, we did not find any differences in phosphorylation levels of IRS2 (data not shown). Since JNK, when activated by oxidative stress, induced serine phosphorylation of IRS1, reducing the insulin signaling pathway (15), protein and phosphorylation levels of JNK were evaluated. We found higher phosphorylation levels of JNK in PRDX62/2 mice compared with WT mice (P , 0.05) (Fig. 2D), suggesting that the reduction of insulin signaling is, at least in part, linked with JNK activation. Next, we measured phosphorylation levels of Akt-1 and Akt-2 at Ser473 and Ser474, respectively, the pyruvate dehydrogenase kinase 2 domains. Phosphorylation of Akt1 and Akt-2 was evident in insulin-stimulated skeletal muscles, and a significant reduction of Akt-1 and Akt-2 phosphorylation levels was found in PRDX62/2 mice compared with WT mice (P , 0.005) (Fig. 2E and F). Since it is known that Akt-2 can modulate GLUT4 cell membrane translocation to increase glucose uptake (16,17), we quantified the amount of membrane-associated GLUT4 after insulin stimulation, showing a significantly lower GLUT4 plasma membrane level in PRDX62/2



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Figure 1—PRDX6 knockout mice are glucose intolerant and insulin resistant. A: Mice IPGTT was performed on 3-month-old male WT (open circle) and knockout (PRDX62/2; filled square) mice weighing 18–20 g. Fasted (16 h) mice received 2 g/kg body weight glucose intraperitoneally, and blood glucose levels were measured at 0, 30, 60, 90, and 120 min after glucose injection. B: ITT was performed on fasted (4 h) WT and PRDX2/2 mice. Mice received an intraperitoneal injection of insulin at 0.75 IU/kg body weight, and blood glucose levels were measured at 0, 15, 30, and 60 min after insulin administration. C: Insulin secretion levels were analyzed by ELISA at 0, 15, 30, and 120 min after glucose injection. D: Insulin gene expression was evaluated on WT mice (white bar) and PRDX62/2 mice (black bar) pancreatic islets. E: Glucose uptake in both mouse models was studied by performing euglycemic-hyperinsulinemic clamp. Values are expressed as mean 6 SE. *P < 0.05, **P < 0.005, ***P < 0.0005. Graphs illustrate one of three separate studies, all yielding similar results (n = 5 mice per group). a.u., arbitrary units; M, mean.

mice compared with WT mice (P , 0.05) (Fig. 2G). Consistent with this finding, the GLUT4 cytoplasmic fraction was significantly higher in the muscle of PRDX62/2 compared with WT mice (P , 0.05) (Fig. 2H), confirming defective transporter translocation by insulin in the mutant strain. Morphological Alteration in Pancreas of PRDX62/2 Mice

A morphometric study was performed to evaluate morphological alterations of pancreatic islets in PRDX62/2 mice. We analyzed five different tissue sections for each animal in the same pancreas area. In Fig. 3A, arrowheads indicate the islets of Langerhans in WT and PRDX62/2 mice. The bar graphs in Fig. 3B and C reveal a reduction in the density and size of islets in PRDX62/2 mice compared with WT mice (P , 0.05), suggesting a relevant role for PRDX6 in maintaining anatomical functional islet mass.

PRDX62/2 Mice Develop Diabetic Dyslipidemia

A dyslipidemic profile characterized by hypertriglyceridemia, high levels of small dense LDL protein, and low levels of HDL cholesterol is often observed in patients with diabetes (18). To gain information on lipid metabolism in PRDX62/2 mice, we measured blood levels of triglycerides, VLDL, HDL cholesterol, and hepatic enzymes. As reported in Table 1, PRDX62/2 mice displayed augmented levels of VLDL and triglycerides and a reduced level of HDL cholesterol compared with WT littermates (P , 0.05), which is like the condition of diabetic dyslipidemia. Moreover, microscopic analysis of liver tissue (Fig. 4A) revealed that PRDX62/2 mice have alterations resembling human NASH, including cell ballooning and lymphocyte infiltration (Fig. 4B). In order to better understand the defects underlying diabetic dyslipidemia, we analyzed the expression of the principal factors regulating lipid metabolism in skeletal

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Figure 2—Western blotting analysis of insulin-signaling pathway. Fasted (overnight) 3-month-old male WT (white bar) and PRDX62/2 (black bar) mice weighing 18–20 g were injected with insulin at 1 IU/kg body weight into the portal vein, and skeletal muscle was collected. One milligram of protein extracts from skeletal muscle was immunoprecipitated with antibodies against IR b-subunit (A) and IRS1 (B) and, after gel separation, was immunoblotted with specific antibodies for phosphotyrosine (PY20 and RC20, respectively). C: PI3K activation was studied by analyzing the interaction between IRS1 and p85 a-subunit. Fifty micrograms of total lysates were immunoblotted with antibodies against JNK (D), Akt1 p-Ser473 (E), and Akt2 p-Ser474 (F). GLUT4 translocation from cytoplasm (H) to plasma membrane (G) was studied by immunoblotting in both mouse models after cellular fractionation. Band intensities were quantified and expressed as mean 6 SE. *P < 0.05 and **P < 0.005 by Student t test. Graphs illustrate one of three separate studies, all yielding similar results (n = 5 mice per group). a.u., arbitrary units; IP, intraperitoneal.

muscle, liver, and WAT (18) (Fig. 5 and Supplementary Table 1 [arrows indicate statistical significance]). In particular, we found a significant increase in PRDX62/2 mice of patatin-like phospholipase domain containing 2 and fatty acid synthase in all three tissues analyzed compared with WT mice, which suggests a possible increase in both lipolytic and lipogenetic processes in these animals. Moreover, in the liver and WAT of PRDX62/2 mice, we observed a significant reduction of peroxisome proliferator– activated receptor g coactivator 1 a, which was not present in the muscle.

PRDX62/2 Mice Develop a Proinflammatory Status

Diabetes is frequently associated with a proinflammatory state that accompanies strong IR (19). Inflammatory status was assessed in PRDX62/2 mice, analyzing the expression of proinflammatory and anti-inflammatory cytokines like interleukin (IL)-6, IL-1b, tumor necrosis factor-a (TNF-a), and IL-10, in liver, skeletal muscle, and WAT. Interestingly, PRDX62/2 mice have a marked increase of IL-1b, TNF-a, and IL-10 in all analyzed tissues (Fig. 6 and Supplementary Table 1 [arrows indicate statistical significance]), while IL-6 was significantly increased in



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Figure 3—Comparative analysis of pancreatic islets in WT and PRDX62/2 mice. Immunohistochemical analysis of pancreatic Langerhans islets (arrowheads) in 3-month-old male WT mice (A, left panel) and PRDX62/2 mice (A, right panel) weighing 18–20 g. Magnification 320. Bar graphs show the quantification of mean islet density (B) and size (C) in PRDX62/2 mice (black bars) compared with WT mice (white bars). Values are expressed as mean 6 SE. *P < 0.05 (n = 5 mice per group).

WAT, but not in skeletal muscle and liver (Fig. 6). Then, we investigated whether chemokine expression levels were increased in PRDX62/2 mice. mRNA expression of chemokine motif ligand 1 was significantly increased in WAT and liver (P , 0.05), but not in skeletal muscle; in contrast, chemokine (C-C motif) ligand 3 mRNA expression was increased in WAT (P , 0.05), liver (P , 0.0005), and skeletal muscle (P , 0.005) (Supplementary Table 1 and Supplementary Fig. 2). These results suggest that Table 1—Metabolic parameters associated with diabetic dyslipidemia WT mice

PRDX62/2 mice

P value

Triglycerides

117 6 14.2

171 6 9.6

,0.05

Cholesterol

109 6 9.5

78 6 6

NS

VLDL

23.4 6 2.8

34.2 6 1.9

,0.05

HDL

65 6 1

52 6 2.6

,0.05

AST

99 6 3.5

106 6 16.6

NS

ALT

45.2 6 8.9

30.7 6 9.6

NS

Data are presented as mean 6 SEM, unless otherwise indicated. ALT, alanine aminotransferase; AST, aspartate aminotransferase.

PRDX62/2 mice develop a proinflammatory status linked with high levels of IR and reduced glucose-stimulated insulin secretion. DISCUSSION

Oxidative stress is considered one of the main mechanisms in the pathogenesis of diabetes and IR (1). Among the antioxidant enzymes that are able to regulate ROSmediated injuries, PRDX6 belongs to a relative new family of proteins that is widely distributed in the tissues of the body (5). PRDX6 is abundant in b-cells (2) as well as in muscle, and its powerful antioxidant role has been reported previously (6). In the current study, we demonstrated that PRDX6 can affect metabolic homeostasis in mice and represents, therefore, a candidate for being a determinant of diabetes susceptibility in humans. We clearly showed that PRDX62/2 mice spontaneously develop a metabolic defect resembling early-stage T2D, which is characterized by higher glucose levels after IPGTT and IR. These defects were accompanied by impaired insulin signaling in the muscle and by reduced insulin secretion in response to glucose. Moreover, we showed a distinctive alteration in lipid profiles and an increase in the inflammatory

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Figure 4—Histological evaluation of liver tissue. A: Representative micrographs showing morphological aspects of NASH as hepatocyte injury or ballooning (arrowheads) and lymphocyte infiltration (arrows) in the liver tissue of 3-month-old male PRDX62/2 mice (right panels) and WT mice (left panels) weighing 18–20 g (magnification 3400). B: Bar graph shows the increased NASH score in PRDX62/2 mice (black bar) compared with WT mice (white bar). Values are expressed as mean 6 SE. **P < 0.005 (n = 5 mice per group).

status of PRDX62/2 mice that are associated with the prediabetic and diabetic phenotypes, which is in agreement with similar findings reported in previous studies (20). Our results are consistent with a pivotal role of PRDX6 in the physiopathology of diabetes and its related complications. To our knowledge, this is some of the first evidence in which modifications of antioxidant defense systems may lead to a diabetic phenotype, even in the absence of direct inducers of oxidative stress. As we described above, PRDX6, compared with other PRDXs, has a double function (phospholipase A2 and peroxidase), which makes it more efficient in the detoxification processes (5). However, other studies are necessary to understand the mechanism of PRDX6 regulation of glucose metabolism and inflammation. It is possible to speculate that PRDX6 is a key enzyme in regulating ROS production, and the action of this enzyme is important even in basal conditions. Indeed, in a model of mice lacking neuronal nitric oxide synthase, PRDX6 upregulation can compensate for the absence of neuronal nitric oxide synthase in scavenging of superoxide; this result supports our hypothesis concerning the importance of oxidative stress unbalance in our model (21). In particular, ROS production has been demonstrated to be important in the etiology of diabetes by acting at the following different levels: 1) blocking glucose uptake (3); 2) impairing insulin signaling pathways (3); 3) altering pancreatic islet morphology (22); 4) modifying metabolic parameters and lipid metabolism (23,24); and 5) increasing inflammatory response (25,26). In the current study, by comparing PRDX62/2 and WT mice, we investigated the role of the enzyme in all of the above mechanisms of ROS-mediated metabolic derangement.

A previous study (27) conducted in a model of knockout mice for SOD1 gene, a potent antioxidant enzyme, demonstrated that mice lacking SOD1 were significantly more susceptible to the development of glucose intolerance, with a nonsignificant reduction of peripheral glucose disposal (;20%). Differently from SOD12/2 mice, PRDX62/2 mice have a significant reduction of peripheral glucose disposal (;31%), suggesting a specific role of PRDX6 in the process leading to IR. Insulin signaling has well-defined pathways, which regulate tissue glucose uptake (28). Interference in each step that regulates these pathways may lead to a decreased uptake of circulating glucose, as depicted in the model in Fig. 7. Mainly, the lack of PRDX6 is associated with a reduction in insulin secretion and a lowering of IRS1 activation in skeletal muscle, leading to reduced levels of Akt-1/Akt-2 phosphorylation, GLUT4 translocation, and then to reduced peripheral glucose uptake. Different levels of IRS1 phosphorylation and activation are key processes that modulate insulin signaling in skeletal muscle (29). The molecules involved in the regulation of oxidative stress have been previously associated with differences in IRS1 activation and expression (30,31). Furthermore, JNK phosphorylation is increased in the skeletal muscle of PRDX62/2 mice, and augmented JNK activation can lead to the deregulation of IRS1, as has already been reported in insulin-resistant nonobese subjects (32). Our results, in agreement with those of previous studies (33,34), support the hypothesis that antioxidant enzymes can reduce insulin-signaling activation and decrease glucose uptake. The morphological changes, including reduction in the size of pancreatic islet mass in mice with diabetes, may



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Figure 5—Genetic expression of the principal enzymes involved in lipid metabolism. Expression levels of genes involved in lipid metabolism, such as patatin-like phospholipase domain containing 2 (PNPLA2), Fas, peroxisome proliferator–activated receptor g coactivator 1 a (PGC1a), and CD36, were analyzed by real-time PCR. mRNA was extracted from skeletal muscle, liver, and WAT of fasted (4 h) WT mice (white bars) and PRDX62/2 mice (black bars). A, E, and I: Increased levels of lipolysis associated with increased lipogenesis in knockout mice (C, G, and M). B, F, and L: Reduction in PGC1a gene expression in PRDX6 mutant mice. D, H, and N: A slight reduction of CD36 expression in liver of PRDX62/2 mice. Values are expressed as mean 6 SE. *P < 0.05, **P < 0.005, ***P < 0.0005 (n = 5 mice per group). a.u., arbitrary units.

result from increased b-cell death and/or birth defects through replication and neogenesis (35). Oxidative stress is an important mechanism leading to these defects (26). We present here evidence that PRDX62/2 mice have a decreased pancreatic b-cell function as suggested by the following: 1) reduction of insulin secretion in response to glucose challenge with higher levels of insulin mRNA expression and 2) decreased area and volume of the islets of Langerhans, probably due to higher levels of apoptotic destruction of pancreatic b-cells, although this latter aspect was not specifically addressed. Interestingly, the overexpression of another PRDX, PRDX4 in mice, has been demonstrated to protect pancreatic b-cells from ROS damage after high-dose streptozotocin-induced diabetes (20). In keeping with the clinical profile of many diabetic patients (36), PRDX62/2 mice have augmented levels of triglycerides and VLDLs and decreased concentrations of HDL. This finding is also in agreement with a potential role of PRDX6 in cardiovascular disease, as previously suggested (37). Furthermore, PRDX62/2 mice had higher NASH scores and liver morphological alterations, features very similar to those present in T2D patients with diabetic dyslipidemia (24). An exacerbated hepatocellular injury was also previously demonstrated in PRDX62/2 mice linked to a different model of hepatic ischemia-reperfusion

injury (38). Moreover, our results are in agreement with previous work, which demonstrated that transgenic diabetic mice with overexpression of PRDX4 had reduced NASH scores and a significant improvement of dyslipidemia (39). In addition, previous results using mice heterozygous for mitochondrial trifunctional protein showed how mitochondrial oxidation may be important in the pathogenesis of nonalcoholic fatty liver disease and NASH associated with IR. This finding provides evidence of the contribution of genetic susceptibility to the development of nonalcoholic fatty liver disease and IR. Furthermore, the authors reported (40) that in the same animal model diets high in polyunsaturated fatty acids can induce NASH. In conclusion, we can hypothesize that PRDXs and genetic susceptibility can have a primary role in the progression of hepatic dysfunction to NASH. PRDX6 has been shown to play an important role during inflammatory processes, and, according to data from a recent study (41), topically administered PRDX6 maintained the homeostasis of corneal cells, reducing inflammation and suppressing neovascularization and apoptosis, induced by ultraviolet irradiation. Inflammation is a main factor leading to diabetes and its associated complications (19). A recent study (42) in b-cells demonstrated that mRNA and protein expression of PRDX6

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Figure 6—Genetic expression of cytokines in insulin target tissues. Expression levels of the main cytokines involved in diabetes and NASH were analyzed by real-time PCR. mRNA was extracted from skeletal muscle, liver, and WAT of fasted (4 h) WT (white bars) and PRDX62/2 (black bars) mice. A, E, and I: Increased levels of IL-6 in WAT of knockout mice. B, F, and L: Higher genetic expression of IL-1b in PRDX62/2 mice. C, G, and M: Increased expression of the anti-inflammatory cytokine IL-10 overall. D, H, and N: TNF-a is highly expressed in PRDX6 mutant mice. Values are expressed as mean 6 SE. *P < 0.05, **P < 0.005, ***P < 0.0005 (n = 5 mice per group). a.u., arbitrary units.

was decreased after treatment with proinflammatory cytokines such as TNF-a and interferon-g. In the current study, we demonstrated a different cytokine profile expression between PRDX62/2 and WT mice, suggesting a relationship between PRDX6 and inflammatory stimuli that would be worth further investigation. Since PRDX-6 can be hyperoxidized and inhibited by excess hydrogen peroxide (43), this enzyme may become inactivated in diabetic patients, thus contributing, in a vicious circle, to disease pathogenesis and progression. Thus, PRDX6 may represent a specific target for antioxidant-based pharmacological interventions; moreover, PRDX6 oxidation level, for instance in blood cells, may represent a novel marker for monitoring oxidative/metabolic status and response to preventive therapy. In line with these ideas, PRDX6 levels were significantly higher in diabetic patients with endothelial dysfunction compared with control subjects, which may possibly represent a physiological adaptation against oxidative stress in patients with atherosclerosis (44). Moreover, another study (45) conducted in diabetic patients demonstrated that physical training was able to increase PRDXs levels measured in erythrocytes, counteracting the oxidative damage that is typical of diabetes. These results are important in outlining the role of the PRDX family in the management of glucose homeostasis and in the prevention of diabetes and cardiovascular disease in diabetic

patients, likely reducing the inflammatory state and oxidative stress. While PRDX62/2 mice represent an excellent model to study the general impact of oxidative stress and reduced antioxidant capacity on glucose homeostasis, we have to acknowledge some limitations of the current study that are typical of studies using transgenic or knockout mice to investigate complex disease (46). These limitations include the following: 1) lack of the evaluation of compensatory mechanisms for the loss of individual proteins and 2) difficulty in distinguishing phenotypes arising from developmental defects from those resulting from impaired signaling. The results of these limitations may be the elevated levels of anti-inflammatory cytokines IL-6 and IL-10 observed in our PRDX62/2 mouse model. However, it is important to remark that the link of IL-6 and IL-10 with IR and diabetes is still controversial. IL-10 has been considered an anti-inflammatory cytokine that improved hepatocellular injury (47); this may explain, at least in part, its increased levels in our animals that were found to have damage from NASH. Genetic variants of PRDX6 have been associated with different responses to chemotherapy in patients with breast cancer because of its antioxidant properties (48). Assessing and comparing PRDX6 expression levels in human skeletal muscle, liver, and/or WAT specimens obtained from control, prediabetic, and frankly diabetic



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laboratory animals. B.C. performed the protein experiments. M.G.S. performed the histological experiments. G.D. and M.T. performed the statistical analysis. A.B. contributed to the analysis of the data and drafting of the manuscript. S.C. and A.C. performed the PCR experiments. F.F. performed the blood analysis. M.F. contributed to the writing of the manuscript. G.S. performed the histological experiments. P.S. conceived the experimental design and contributed to the writing of the manuscript. D.D.-M. helped to organize the data and contributed to the writing of the manuscript. A.G. handled the laboratory animals and contributed to the data analysis. A.O. conceived the histological protocols and contributed to the data analysis. D.L. helped to conceive the experimental design and organize the data, and contributed to the writing of the manuscript. D.L. 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. Prior Presentation. Parts of this study were presented in abstract form at the 74th Scientific Sessions of the American Diabetes Association, San Francisco, CA, 13–17 June 2014.

References

Figure 7—Schematic illustration of PRDX62/2-impairing insulin signaling pathways in skeletal muscle. PRDX62/2 impairs insulin signaling pathways in skeletal muscle at different levels, as follows: 1) decrease of insulin secretion by b-cells; 2) reduction of IRS1 tyrosine phosphorylation with subsequent diminished interaction between IRS1 and PI3K; 3) decrease of PKB/Akt phosphorylation and activation; 4) lower GLUT4 translocation from cytoplasmic reserve pool to plasma membrane; and 5) less glucose uptake. P, phosphorylation.

individuals in association with genetic studies evaluating polymorphisms of the PRDX6 gene would have great impact on further understanding the role of PRDX6 in the severity of inflammatory conditions such as diabetes. In conclusion, on the basis of the present data, the emerging picture is of a complex interaction between PRDX6 and insulin-signaling pathways involved in the regulation of glucose homeostasis, lipid metabolism, and inflammatory response. PRDX6, through its functional activity against oxidative stress and inflammation, may be indicated as a new molecular target that will be useful in the development of preventive strategies and novel therapies for T2D and its related diseases. Further studies should be performed to clarify and better define the role of PRDX6 in diabetes and its associated metabolic dysfunctions. Funding. This work was supported by Research Project 2009 grant, Fondazione Roma; PRIN 2010 and 2011 grants from the Ministero dell’Istruzione, dell’Università e della Ricerca (to D.L. and P.S.); Fondazione Umberto Di Mario; Società Italiana Diabetologia (SID) Lazio grant; 2010 Grant from Associazione Italiana per la Ricerca sul Cancro; and grant D.3.2-2013 from Università Cattolica del Sacro Cuore. Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. F.P. and R.A. performed laboratory experiments and contributed to the writing of the manuscript. G.P.S. and D.P. handled the

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