Diabetes
1
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
DOI: 10.2337/db14-0144
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 knock-out 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 (C57/BL6: 129
1Department
Corresponding author: Davide Lauro, [email protected].
of System Medicine, University of Rome Tor Vergata, Italy
of Endocrinology and Metabolic Diseases, Università Cattolica del Sacro
Cuore, Rome, Italy
3Diabetic Care Clinics, Associazione Cavalieri Italiani Sovrano Militare Ordine Di
Malta, Rome, Italy
4Anatomic Pathology, Department of Biomedicine and Prevention, University of
Rome Tor Vergata, 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 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 Publish Ahead of Print, published online June 19, 2014
METABOLISM
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 knock-out
(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.
2
PRDX6 and Pathogenesis of Diabetes
SvJ) were provided by Professor Xiaosong Wang (The Jackson
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 One Touch 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.
Diabetes
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 membranes 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 Trans Blot Turbo Transfer System (Bio-Rad
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), Akt-1, Akt-2, and stressactivated 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 (BioRad 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
diabetes.diabetesjournals.org
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 203 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 realtime 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 the 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
Pacifici and Associates
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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 Serine473 and Serine474, respectively, the
pyruvate dehydrogenase kinase 2 domains. Phosphorylation of Akt-1 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
4
PRDX6 and Pathogenesis of Diabetes
Diabetes
Figure 1—PRDX6 knock-out mice are glucose intolerant and insulin resistant. A: Mice IPGTT was performed on 3-month-old male WT
(open circle) and knock-out (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 (open circle) and 2/2 (filled square). 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 (WT mice, open circle; PRDX62/2 mice, filled square). 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 (WT mice,
white bar; PRDX62/2 mice, black bar) was studied by performing euglycemic-hyperinsulinemic clamp. Values are expressed as the 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 PRDX6 2/2 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 the mean 6 SE.
*P < 0.05, **P < 0.005, ***P < 0.0005 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 [where arrows indicate statistical significance]), while IL-6 was significantly increased
6
PRDX6 and Pathogenesis of Diabetes
Diabetes
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 the mean 6 SE. *P < 0.05 (n = 5 mice per group). (A high-quality color representation of this figure is
available in the online issue.)
in 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
Table 1—Metabolic parameters associated with diabetic
dyslipidemia
P value
Parameters
WT mice
PRDX62/2 mice
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
,0.05
HDL
65 6 1
52 6 2.6
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 the mean 6 SEM, unless otherwise
indicated. ALT, alanine aminotransferase; AST, aspartate
aminotransferase.
and Supplementary Fig. 2). These results suggest that
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
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Figure 4—Histological evaluation of liver tissue. 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. A: 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 the mean 6 SE. **P < 0.005 (n = 5 mice per group). (A high-quality color
representation of this figure is available in the online issue.)
showed a distinctive alteration in lipid profiles and an
increase in the inflammatory 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.
8
PRDX6 and Pathogenesis of Diabetes
Diabetes
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, C, E, G, I, and M: Increased levels of lipolysis associated with increased lipogenesis in
knockout mice. 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 the mean 6 SE. *P < 0.05, **P < 0.005, ***P < 0.0005 (n = 5 mice per
group). a.u., arbitrary units.
The morphological changes, including reduction in the
size of pancreatic islet mass in mice with diabetes, may
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 very low-density proteins (i.e., 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
diabetes.diabetesjournals.org
Pacifici and Associates
9
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.
complications (19). A recent study (42) in b-cells demonstrated that mRNA and protein expression of PRDX6
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 knock-out 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
10
PRDX6 and Pathogenesis of Diabetes
Diabetes
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
laboratory animals. B.C. performed the protein experiments. M.G.S. performed
the histological experiments. G.D. and M.T. performed the statistical analysis.
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.
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. (A high-quality color representation of this figure is available in the online issue.)
human skeletal muscle, liver, and/or WAT specimens
obtained from control, prediabetic, and frankly diabetic
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.
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