Vol.3, No.3, 101-110 ( 2013) Journal of Diabetes Mellitus
http://dx.doi.org/10.4236/jdm.2013.33015
12/15-Lipoxygenase inhibition counteracts MAPK
phosphorylation in mouse and cell culture models
of diabetic peripheral neuropathy
Roman Stavniichuk1, Alexander A. Obrosov1, Viktor R. Drel1, Jerry L. Nadler2,
Irina G. Obrosova1, Mark A. Yorek3*
1Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, USA
2Department of Internal Medicine, Eastern Virginia Medical School, Norfolk, USA
3Department of Veterans Affairs Iowa City Health Care System and Department of Internal Medicine, University of Iowa, Iowa City,
USA; *Corresponding Author:
Received 14 May 2013; revised 16 June 2013; accepted 23 June 2013
Copyright © 2013 Roman Stavniichuk et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Background: Increased mitogen-activated pro-
tein kinase (MAPK) phosphorylation has been
detected in peripheral nerve of human subjects
and animal models with diabetes as well as
high-glucose exposed human Schwann cells,
and have been implicated in diabetic peripheral
neuropathy. In our recent studies, leukocyte-
type 12/15-lipoxygenase inhibition or gene defi-
ciency alleviated large and small nerve fiber
dysfunction, but not intraepidermal nerve fiber
loss in streptozotocin-diabetic mice. Methods:
To address a mechanism we evaluated the po-
tential for pharmacological 12/15-lipoxygenase
inhibition to counteract excessive MAPK phos-
phorylation in mouse and cell culture models of
diabetic neuropathy. C57Bl6/J mice were made
diabetic with streptozotocin and maintained
with or without the 12 /1 5-lipoxygenase inhibitor
c innamyl-3,4-dihydroxy-α-cyanocinnamate (CDC).
Human Schwann cells were cultured in 5.5 mM
or 30 mM glucose with or without CDC. Results:
12(S) HETE concentrations (ELISA), as well as
12/15-lipoxygenase expression and p38 MAPK,
ERK, and SAPK/JNK phosphorylation (all by
Western blot analysis) were increased in the
peripheral nerve and spinal cord of diabetic
mice as well as in high glucose-exposed human
Schwann cells. CDC counteracted diabetes-in-
duced increase in 12(S)HETE concentrations (a
measure of 12/15-lipoxygenase activity), but not
12/15-lipoxygenase overexpression, in sciatic
nerve and spinal cord. The inhibitor blunted
excessive p38 MAPK and ERK, but not SAPK/
JNK, phosphorylation in sciatic nerve and high
glucose exposed human Schwann cells, but did
not affect MAPK, ERK, and SAPK/JNK phos-
phorylation in spinal cord. Conclusion: 12/15-
lipoxygenase inhibition counteracts diabetes
related MAPK phosphorylation in mouse and
cell culture models of diabetic neuropathy and
implies that 12/15-lipoxygenase inhibitors may
be an effective treatment for diabetic peripheral
neuropathy.
Keywords: Diabetes; Lipoxygenase; Neuropathy;
Schwann Cells; Mitogen -Activated Protein Kinase
1. INTRODUCTION
Diabetic peripheral neuropathy (DPN), affects at least
50% of patients with both Type 1 and Type 2 diabetes,
and is a leading cause of foot amputation [1-3]. DPN is
manifested by motor (MNCV) and sensory (SNCV)
nerve conduction velocity deficits as well as microvas-
cular dysfunction and by increased vibration and thermal
perception thresholds that progress to sensory loss, oc-
curring in conjunction with degeneration of all fiber
types in the peripheral nerve [1-4]. A significant propor-
tion of patients with DPN also describe abnormal sensa-
tions such as paresthesias, allodynia, hyperalgesia, and
spontaneous pain [3]. The pathogenesis of DPN has ex-
tensively been studied in animal and cell culture models.
Multiple biochemical changes have been attributed to the
etiology of diabetic neuropathy including, but not limited
to, increased activity of the sorbitol pathway [5-7], gen-

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110
102
eration of methylglyoxal [8,9] and non-enzymatic glyca-
tion/glycoxidation [10,11], oxidative-nitrosative stress
[12-16], impaired neurotrophic support [17,18], and ac-
tivation of protein kinase C [19,20], poly(ADP-ribose)
polymerase [21,22], and of the enzymes of arachidonic
acid metabolism, cyclooxygenase-2 [23] and leukocyte-
type 12/15-lipoxygenase [24,25]. Increased mitogen-
activated protein kinase (MAPK) phosphorylation was
detected in peripheral nerve of human subjects with dia-
betes [26], several animal models of diabetes [26-31],
and high-glucose exposed cultured human Schwann cells
[32], and has been implicated in the pathophysiology of
diabetic peripheral neuropathy.
Multiple specific small molecule MAPK inhibitors are
now in clinical trials for chronic diseases including sev-
eral types of cancer, inflammatory and autoimmune dis-
eases, neuropathic pain following nerve trauma, as well
as Parkinson’s and Alzheimer’s diseases [33]. Regardless
of these efforts and outcomes, the search for alternative
approaches to inhibit excessive MAPK phosphorylation
in specific pathological conditions including diabetic
neuropathy is highly warranted. In our previous experi-
ments [24,25,31], 12/15-lipoxygenase inhibition and
gene deficiency improved several diabetic neuropathy
associated endpoints in streptozotocin-diabetic mice in-
cluding nerve conduction deficits and behavioral changes
suggesting that preventing 12/15-lipoxygenase activation
may be an effective treatment for diabetic neuropathy.
2. MATERIALS AND METHODS
2.1. Reagents
Unless otherwise stated, all chemicals were of re-
agent-grade quality, and were purchased from Sigma
Chemical Co., St. Louis, MO, USA. Cinnamyl-3,4-
dihydroxy-alpha-cyanocinnamate (CDC) was obtained
from Enzo Life Sciences International, Plymouth Meet-
ing, PA, USA. For Western blot analyses in mouse tis-
sues, rabbit polyclonal (clone H-100) anti-12-lipoxy-
genase (LO) antibody, rabbit polyclonal (clone H-147)
anti-p38 MAPK antibody, mouse monoclonal (clone
MK1) anti-ERK antibody, and rabbit polyclonal (clone
C17) anti-JNK1 antibody were obtained from Santa Cruz
Biotechnology, Santa Cruz, CA, USA. Rabbit polyclonal
anti-phospho-p38 MAPK antibody, rabbit monoclonal
(clone D13.14.4E) anti-phospho-ERK antibody, and rab-
bit polyclonal anti-phospho-SAPK/JNK antibody were
purchased from Cell Signaling Technology, Boston, MA,
USA. For Western blot analyses in human Schwann cells,
rabbit polyclonal (clone C16) antibody against total ERK
and mouse monoclonal (clone E4) antibody against
phosphorylated ERK were obtained from Santa Cruz
Biotechnology, Santa Cruz, CA. For other MAPKs, the
antibodies listed above were used.
2.2. Animals
The experiments were performed in accordance with
The Guide for the Care and Handling of Laboratory
Animals (NIH Publication No. 85-23) and Pennington
Biomedical Research Center Protocol for Animal Studies.
Mature male C57Bl6/J mice were purchased from Jack-
son Laboratories. All the mice were fed standard mouse
chow (PMI Nutrition International, Brentwood, MO,
USA) and had ad libitum access to water. After a 7-day
acclimation in a new environment, the mice were ran-
domly divided into two groups. In one group, diabetes
was induced by streptozotocin (STZ) as we described
previously [24,25]. The mice with blood glucose ≥13.8
mM, three days post streptozotocin were considered dia-
betic. The control and diabetic mice were kept for 12
weeks without treatment, and then divided into two sub-
groups that were maintained with or without treatment
with cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC),
8 mg kg/d, s.c., for another 4 weeks. CDC, at the afore-
mentioned dose, counteracted multiple manifestations of
diabetic neuroapthy and oxidative-nitrosative stress in
peripheral nerve and spinal cord in our previous study
[24]. Non-fasting blood glucose measurements were
performed after induction of diabetes and at the end of
the study period.
2.3. Anesthesia, Euthanasia and Tissue
Sampling
The animals were sedated by CO2, and immediately
sacrificed by cervical dislocation. Sciatic nerves and
spinal cords were rapidly dissected and frozen in liquid
nitrogen for subsequent assessment of LO as well as total
and phosphorylated p38 MAPK, ERK, and SAPK/JNK
levels, and 12(S)-HETE concentrations.
2.4. Human Schwann Cell Culture
Schwann cells play a key role in the pathology of
various inflammatory, metabolic, and hereditary poly-
neuropathies, including diabetic neuropathy [34,35]. Pre-
vious studies demonstrated that cultured human Schwann
cells (cell line cat. #1700, ScienCell, Carlsbad, CA)
manifest increased superoxide production, accumulation
of nitrated and poly(ADP-ribosyl)ated proteins and 4-
hydroxynonenal adducts, inducible nitric oxide synthase
overexpression, 12/15-Lipoxygenase overexpression and
activation, increased p38 MAPK phosphorylation, down-
regulation of taurine transporter, as well as impaired in-
sulin signaling early (1 - 7 d) after exposure to high glu-
cose [24,36-38]. They therefore represent a good model
for studying interactions among individual pathobio-
chemical mechanisms in the peripheral nerve. In the
present study, human Schwann cells (passages 7 - 10)
were cultured in 6-well plates in media containing 5.5

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110 103
mM D-glucose. At ~70% confluence, the media were
replaced with those containing either 5.5 mM D-glucose
or 30 mM D-glucose with or without CDC, 10 M (6 - 8
plates per condition). After 24 hr, the cells were used for
assessment of total and phosphorylated p38 MAPK,
ERK, and SAPK/JNK.
2.5. Specific Methods
2.5.1. Western Bl ot A nalyses of LO and Total
and Phosphorylated p38 MAPK, ERK,
and SAPK/JNK
Sciatic nerve and spinal cord materials (~3 - 10 mg) or
scraped human Schwann cells were placed on ice in 100
µL of buffer containing 50 mmol/l Tris-HCl, pH 7.2; 150
mmol/l NaCl; 0.1% sodium dodecyl sulfate; 1% NP-40;
5 mmol/l EDTA; 1 mmol/l EGTA; 1% sodium deoxy-
cholate and the protease/ phosphatase inhibitors leu-
peptin (10 μg/ml), pepstatin (1 μg/ml), aprotinin (20 μg/
ml), benzamidine (10 mM), phenylmethylsulfonyl fluo-
ride (1 mM), sodium orthovanadate (1 mmol/l), and ho-
mogenized on ice. The homogenates were sonicated and
centrifuged at 14,000 g for 20 min. All the afore-men-
tioned steps were performed at 4˚C. The lysates (20 μg
protein for sciatic nerve and 40 μg for spinal cord and
human Schwann cells) were mixed with equal volumes
of 2x sample-loading buffer containing 62.5 mmol/l Tris-
HCl, pH 6.8; 2% sodium dodecyl sulfate; 5% -mer-
captoethanol; 10% glycerol, and 0.025% bromophenol
blue, and fractionated in 10 % (total and phosphorylated
MAPKs) or 7.5% (lipoxygenase) SDS-PAGE in an elec-
trophoresis cell (Mini-Protean III; Bio-Rad Laboratories,
Richmond, CA). Electrophoresis was conducted at 15
mA constant current for stacking, and at 25 mA for pro-
tein separation. Gel contents were electrotransferred (80
V, 2 hr) to nitrocellulose membranes using Mini Trans-
Blot cell (Bio-Rad Laboratories, Richmond, CA) and
Western transfer buffer (10X Tris/Glycine buffer, Bio-
Rad Laboratories, Richmond, CA) diluted with 20% (v/v)
methanol. Free binding sites were blocked in 5% (w/v)
BSA in 20 mmol/l Tris-HCl buffer, pH 7.5, containing
150 mmol/l NaCl and 0.05% Tween 20, for 1 h. Primary
antibodies against 12/15-lipoxygenase, or phosphory-
lated p38 MAPK, ERK, or SAPK/JNK were applied at
4˚C overnight, after which secondary antibodies were
applied at room temperature for 1 h. After extensive
washing, protein bands detected by the antibodies were
visualized with the Amersham ECLTM Western Blotting
Detection Reagent (Little Chalfont, Buckinghamshire,
UK). Membranes previously probed for phosphorylated
MAPKs were then stripped in the 25 mmol/l glycine-HCl
buffer, pH 2.5, containing 2% SDS, and reprobed with
antibodies against total p38 MAPK, ERK, and SAPK/
JNK, respectively. Membranes previously probed for 12/
15-lipoxygenase were stripped again and reprobed with
β-actin antibody to confirm equal protein loading.
2.5.2. ELISA 12(S)HETE Measurements
For assessment of 12(S)HETE, sciatic nerve and spi-
nal cord samples were homogenized on ice in 15 mM
Tris-HCI buffer (1:100 w/v) containing 140 mM NaCl,
pH 7.6, and centrifuged. 12(S)HETE was measured in
supernatants with the 12(S)-hydroxyeicosatetraenoic acid
[12(S)HETE] Enzyme Immuno Assay kit (Assay De-
signs, Ann Arbor, MI) according to manufacturer’s in-
structions.
2.6. Statistical Analysis
The results are expressed as Mean ± SEM. Data were
subjected to equality of variance F test, and then to log
transformation, if necessary, before one-way analysis of
variance. Where overall significance (p < 0.05) was at-
tained, individual between group comparisons for multi-
ple groups were made using the Student-Newman-Keuls
multiple range test. When between-group variance dif-
ferences could not be normalized by log transformation
(datasets for body weights and plasma glucose), the data
were analyzed by the nonparametric Kruskal-Wallis one-
way analysis of variance, followed by the Bonferroni/
Dunn test for multiple comparisons. Individual pair-wise
comparisons in experiments 3 and 4 were made using the
unpaired two-tailed Student’s t-test or Mann-Whitney
rank sum test where appropriate. Significance was de-
fined at p < 0.05.
3. RESULTS
3.1. Animal Experiments
3.1.1. Body Weights and Blood Glucose
Concentrations
The initial (prior to streptozotocin administration)
body weights were similar in all experimental groups
(Table 1). Weight gain during the 16-wk study was lower
in both untreated and CDC-treated diabetic mice than in
the non-diabetic control group. CDC treatment did not
affect weight gain in either control or diabetic mice. Ini-
tial (after streptozotocin administration) non-fasting
blood glucose concentrations were 2.0-fold and 1.9-fold
higher in untreated and CDC-treated diabetic mice than
in the control group. Hyperglycemia progressed with the
prolongation of diabetes, and the differences between
final blood glucose concentrations in both diabetic groups
and non-diabetic controls exceeded 3-fold. CDC treat-
ment did not affect non-fasting blood glucose concentra-
tions in either non-diabetic or diabetic mice.
3.1.2. 12/15-Lipoxygenase Expression and
12(S)HETE Concentrations
Sciatic nerve (Figures 1(a) and (b)) and spinal cord

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110
104
(Figures 1(d) and (e)) 12/15-lipoxygenase expression
was increased by 40% and 57%, respectively, in diabetic
mice compared with controls. CDC treatment did not af-
fect sciatic nerve and spinal cord 12/15-lipoxygenase ex-
pression in either control or diabetic mice. Sciatic nerve
(Figure 1(c)) and spinal cord (Figure 1(f)) 12(S)HETE
concentrations, a measure of 12/15-lipoxygenase ac-
tivity, were increased by 223% and 48%, respectively,
in diabetic mice compared with controls. CDC treatment
blunted diabetes-associated sciatic nerve and spinal cord
12(S)HETE accumulation.
3.1.3. MAPK Expression in Sciatic Nerve and
Spinal Cord
Diabetic wild-type mice displayed 108%, 35%, and
56% increases in sciatic nerve p38 MAPK (Figures 2(a),
Ta b le 1 . Initial and final body weights and blood glucose concentrations in control and diabetic mice maintained with and without
CDC inhibitor treatment.
Body weight (g) Blood glucose (mmol/l)
Variable Group
Initial Final Initial Final
Control 24.6 ± 0.4 35.6 ± 1.2 8.2 ± 0.6 8.4 ± 0.2
Control + CDC 25.1 ± 0.3 35.3 ± 1.3 8.6 ± 0.3 8.3 ± 0.4
Diabetic 25.3 ± 0.5 27.9 ± 0.6* 16.4 ± 1.0* 25.6 ± 1.4*
Diabetic + CDC 25.2 ± 0.6 27.2 ± 0.5* 16.0 ± 1.2* 27.1 ± 1.6*
Data are expressed as Means ± SEM. n = 15 per group. *p < 0.01 vs non-diabetic control group.
Figure 1. Representative Western blot analyses of 12/15-lipoxygenase expression (a), (d), 12/15-lipoxygenase protein contents (den-
sitometry, %, (b), (e)), and 12(S)HETE concentrations (c), (f) in the sciatic nerve and spinal cord of non-diabetic control and diabetic
mice maintained with or without cinnamyl-3,4-dihydroxy-α-cyanocinnamate treatment. C-control; D-diabetic; CDC-cinnamyl-3,4-
dihydroxy-α-cyanocinnamate. Mean ± SEM, n = 6 - 11 per group. *, **p < 0.05 and <0.01 vs non-diabetic control group. #, ## p < 0.05
nd <0.01 vs untreated diabetic group. a
OPEN A CCESS

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110 105
(b)), ERK (Figures 2(d), (e)), and SAPK/JNK (Figures
2(g), (h)) phosphorylation, compared with the corre-
sponding control group. Total p38 MAPK (Figures 2(a),
(c)), ERK (Figures 2(d), (f)), and SAPK/JNK (Figures
2(g), (i)) levels were indistinguishable among the groups.
CDC treatment did not affect the phosphorylation state of
any of three MAPKs in non-diabetic mice. It reduced
p38 and ERK phosphorylation by 58% and 23% (p <
0.05 vs corresponding untreated group for both com-
parisons), but did not affect SAPK/JNK phosphorylation,
in diabetic mice. Spinal cord p38 MAPK (Figures 3(a),
(b)), ERK (Figures 3(d), (e)) and SAPK/JNK (Figures
3(g), (h)) phosphorylation was elevated in both diabetic
untreated mice, compared with the non-diabetic controls.
Total p38 MAPK (Figures 3(a), (c)), ERK (Figures 3(d),
(f)), and SAPK/JNK (Figures 3(g), (i)) levels were simi-
lar in control and diabetic mice maintained with or
without CDC treatment. The 12/15-lipoxygenase inhibi-
tor did not affect the phosphorylation state of any of
three MAPKs in either non-diabetic or diabetic mice.
3.2. Human Schwann Cells Experiment
MAPK Expression
Phosphorylation of p38 MAPK (Figures 4(a), (b)),
ERK (Figures 4(d), (e)), and SAPK/JNK (Figures 4(g),
(h)) was increased by 47%, 38%, and 95% in human
Schwann cells cultured in 30 mM glucose, compared
with those cultured in 5.5 mM glucose. Total p38 MAPK
(Figure 4(c)), ERK (Figure 4(d)), and SAPK/JNK (Fig-
ure 4(i)) levels were indistinguishable among the ex-
perimental groups. CDC treatment did not affect levels
of any total and phosphorylated MAPKs in human
Schwann cells cultured in 5.5 mM glucose, but com-
pletely prevented high glucose-induced increase in hu-
man Schwann cells p38 MAPK and ERK, but not SAPK/
JNK, phosphorylation.
Figure 2. Representative Western blot analyses of phosphorylated and total p38 MAPK, ERK, and SAPK/JNK expression (a), (d),
(g), and phosphorylated (b), (e), (h) and total (c), (f), (i) p38 MAPK, ERK, and SAPK/JNK protein contents (densitometry, %) in the
sciatic nerve of non-diabetic control and diabetic mice maintained with or without cinnamyl-3,4-dihydroxy-α-cyanocinnamate treat-
ment. C-control; D-diabetic; CDC-cinnamyl-3,4-dihydroxy-α-cyanocinnamate. Mean ± SEM, n = 8 - 9 per group. *, **p < 0.05 and
<0.01 vs non-diabetic control group. #, ##p < 0.05 and <0.01 vs untreated diabetic group.

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110
106
Figure 3. Representative Western blot analyses of phosphorylated and total p38 MAPK, ERK, and SAPK/JNK expression (a), (d),
(g), and phosphorylated (b), (e), (h) and total (c), (f), (i) p38 MAPK, ERK, and SAPK/JNK protein contents (densitometry, %) in the
spinal cord of non-diabetic control and diabetic mice maintained with or without cinnamyl-3,4-dihydroxy-α-cyanocinnamate treat-
ment. C-control; D-diabetic; CDC-cinnamyl-3,4-dihydroxy-α-cyanocinnamate. Mean ± SEM, n = 6-8 per group. *, **p < 0.05 and
<0.01 vs non-diabetic control group.
4. DISCUSSION
The findings described herein indicate that pharma-
cological inhibition of 12/15-lipoxygenase suppresses
diabetes-induced excessive p38 MAPK and ERK, but not
SAPK/JNK, phosphorylation in mouse sciatic nerve.
Furthermore, 12/15-lipoxygenase inhibition blunts high
glucose-induced p38 MAPK and ERK, but not SAPK/
JNK, phosphorylation in human Schwann cells, thus sug-
gesting the existence of the similar relationship be-
tween 12/15-lipoxygenase and MAPK in diabetic pe-
ripheral neuropathy in humans.
Evidence for the important role of the enzymes of
arachidonic acid metabolism, cyclooxygenase-2 (COX-2,
[23,39]) and 12/15-lipoxygenase [24,25], in functional,
morphological, and biochemical abnormalities in dia-
betic peripheral neuropathy is emerging. Increased acti-
vity of COX-2 was implicated in motor nerve conduction
velocity and sensory nerve conduction velocity deficits,
oxidative stress, and inflammation associated with ex-
perimental diabetic peripheral neuropathy [23,39], as
well as in diabetic cardiac autonomic neuropathy and left
ventricular dysfunction [40]. In our previous studies [24,
25], increased activity of 12/15-lipoxygenase was identi-
fied as an important contributor to diabetes-induced mo-
tor and sensory nerve conduction slowing, thermal and
mechanical hypoalgesia, axonal atrophy of large myeli-
nated fibers, and oxidative-nitrosative stress in peripheral
nerve and spinal cord. Despite its clear role in oxidative-
nitrosative stress, 12/15-lipoxygenase activation was not
involved in diabetes-associated reduction in intraepider-
mal nerve fiber density [24,25]. This makes exploration
of the protective and pathobiochemical processes trig-
gered through 12/15-lipoxygenase in tissue-sites for dia-
betic peripheral neuropathy particularly interesting.
Studies in animal models of diabetes-associated athero-
sclerosis and in vascular smooth muscle cells exposed to

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110 107
Figure 4. Representative Western blot analyses of phosphorylated and total p38 MAPK, ERK, and SAPK/JNK expression (a), (d),
(g), and phosphorylated (b), (e), (h) and total (c), (f), (i) p38 MAPK, ERK, and SAPK/JNK protein contents (densitometry, %) in
human Schwann cells cultured in normal (5.5 mM) or high (30 mM) glucose with or without cinnamyl-3,4-dihydroxy-α-cyanocin-
namate. C-control; D-diabetic; CDC-cinnamyl-3,4-dihydroxy-α-cyanocinnamate. Mean ± SEM, n = 5 - 7 per group. *, **p < 0.05 and
<0.01 vs cells cultured in normal glucose. ##p < 0.01 vs cells cultured in high glucose without CDC.
the diabetic milieu [41-45] revealed that 12/15-lipoxy-
genase overexpression and activation is implicated in
multiple biochemical changes including increased phos-
phorylation of ERK and p38 MAPK, Ras activation,
cAMP response element-binding protein (CREB) phos-
phorylation, DNA-binding activity, and transactivation,
overexpression of intercellular adhesion molecule-1,
monocyte chemoattractant protein-1, and interleukin-6,
activation of nuclear factor- B, Src tyrosine kinase, fo-
cal adhesion kinase, and Akt, and histone H3-Lys-9/14
acetylation. The important role for 12/15-lipoxygenase in
increased p38 MAPK, ERK, and CREB phosphorylation,
and increased activator protein-1 and CREB DNA bind-
ing and transcriptional activities, as well as fibronectin
overexpression has been identified in renal mesangial
cells isolated from diabetic mice [46]. We have been par-
ticularly interested in the relationship between 12/15-
lipoxygenase and MAPKs, because both p38 MAPK
[26-28,47-49] and, recently, ERK [50,51], have been
implicated in neuropathic changes in diabetes. In par-
ticular, increased p38 MAPK phosphorylation is in-
volved in nerve conduction deficit [27], diabetic erectile
autonomic neuropathy and vasculopathy [47], mechani-
cal hyperalgesia [28,48], and tactile allodynia [49]. In-
creased ERK phosphorylation contributes to tactile allo-
dynia [50] and mechanical hyperalgesia [51]. Further-
more, inhibition of p38 MAPK with its specific inhibit-
tors SB239063 and SB203580 counteracted diabetes-
associated GSH depletion in the peripheral nerve and
COX-2, inducible nitric oxide synthase, and tumor ne-
crosis factor- overexpression in dorsal root ganglion
neurons [28,52]. Our recent experiments [31] revealed
that 12/15-lipoxygenase gene deficiency prevents dia-
betes-associated excessive p38 MAPK, ERK, but not
SAPK/JNK phosphorylation, in peripheral nerve, and
p38 MAPK, ERK, and SAPK/JNK phosphorylation in

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110
108
DRG. Interestingly, spinal cord p38 MAPK, ERK, and
SAPK/JNK phosphorylation induced by diabetes, ap-
peared independent of the LO mechanism. In the present
study, similar findings in peripheral nerve and spinal
cord were obtained with the 12/15-lipoxygenase inhibitor
CDC. This is consistent with our previous results [24,25]
suggesting that CDC, often considered as a relatively
weak and non-specific12/15-lipoxygenase inhibitor [53-
55], can be used to dissect the role for leukocyte-type
12/15-lipoxygenase in diabetic peripheral neuropathy,
and that the role for 12/15-lipoxygenase in MAPK acti-
vation varies in different tissues-12/15-lipoxygenase
products have been found to cause SAPK/JNK activation
in fibroblasts [56], but, apparently, this mechanism does
not mediate high glucose-induced SAPK/JNK activation
in human Schwann cells.
In conclusion, the present pharmacological study dis-
sects the role for12/15-lipoxygenase in diabetes- and
high glucose-induced MAPK activation in tissue-sites for
diabetic peripheral neuropathy and human Schwann cells.
They support interaction between the two mechanisms in
the peripheral nerve, but not in spinal cord. The findings
are relevant to understanding the mechanisms of diabetic
peripheral neuropathy in humans, and suggest, that to-
gether with MAPK inhibitors, 12/15-lipoxygenase in-
hibitors can be used as pharmacological tool for inhibit-
ing excessive MAPK phosphorylation in experimental,
and, potentially, future clinical studies. In addition, our
findings suggest that the 12/15-lipoxygenase inhibitor
CDC can be used for dissection of the pathobiochemical
mechanisms triggered by leukocyte-type 12/15-lipoxy-
genase, because the biochemical effects of CDC closely
mimic those of leukocyte-type 12/15-lipoxygenase gene
deficiency.
5. ACKNOWLEDGEMENTS
The study was supported in part by the National Institutes of Health
Grants DK074517 (to I.G.O.), DK077141 (to I.G.O. and M.A.Y.),
DK081147 (to I.G.O. and M.A.Y.), DK073990 (to M.A.Y.) and the
American Diabetes Association Research Grant 7-08-RA-102 (to I.G.O.).
The authors thank Dr.Rama Natarajan for valuable help with antibodies
selections.
REFERENCES
[1] Boulton, A.J., Vinik, A.I., Arezzo, J.C., Bril, V., Feldman,
E.L., Freeman, R., et al. (2005) Diabetic neuropathies: A
statement by the American Diabetes Association. Diabe-
tes Care, 28, 956-962. doi:10.2337/diacare.28.4.956
[2] Sinnreich, M., Taylor, B.V. and Dyck, P.J. (2005) Diabetic
neuropathies. Classification, clinical features, and patho-
physiological basis. Neurologist, 11, 63-79.
doi:10.1097/01.nrl.0000156314.24508.ed
[3] Veves, A., Backonja, M. and Malik, R.A. (2008) Painful
diabetic neuropathy: Epidemiology, natural history, early
diagnosis, and treatment options. Pain Medicine, 9, 660-
674. doi:10.1111/j.1526-4637.2007.00347.x
[4] Tesfaye, S., Boulton, A.J., Dyck, P.J., Freeman, R., Horo-
witz, M., Kempler, P., et al. (2010) Diabetic neuropathies:
Update on definitions, diagnostic criteria, estimation of
severity, and treatments. Diabetes Care, 33, 2285-2293.
doi:10.2337/dc10-1303
[5] Yagihashi, S., Yamagishi, S.I., Wada, R.R., Baba, M.,
Hohman, T.C., Yabe-Nishimura, C., et al. (2001) Neu-
ropathy in diabetic mice overexpressing human aldose
reductase and effects of aldose reductase inhibitor. Brain,
124, 2448-2458. doi:10.1093/brain/124.12.2448
[6] Obrosova, I.G., Van Huysen, C., Fathallah, L., Cao, X.C.,
Greene, D.A. and Stevens, M.J. (2002) An aldose reduc-
tase inhibitor reverses early diabetes-induced changes in
peripheral nerve function, metabolism, and antioxidative
defense. The FASEB Journal, 16, 123-125.
[7] Ho, E.C., Lam, K.S., Chen, Y.S. and Yip, J.C., Arvindak-
shan, M., Yamagishi, S., et al. (2006) Aldose reductase-
deficient mice are protected from delayed motor nerve
conduction velocity, increased c-Jun NH2-terminal kinase
activation, depletion of reduced glutathione, increased
superoxide accumulation, and DNA damage. Diabetes, 55,
1946-1953. doi:10.2337/db05-1497
[8] Jack, M.M., Ryals, J.M. and Wright, D.E. (2011) Charac-
terization of glyoxalase I in a streptozocin-induced mouse
model of diabetes with painful and insensate neuropathy.
Diabetologia, 54, 2174-2182.
doi:10.1007/s00125-011-2196-3
[9] Bierhaus, A., Fleming, T., Stoyanov, S., Leffler, A., Babes,
A., Neacsu, C., et al. (2012) Methylglyoxal modification
of Na(v)1.8 facilitates nociceptive neuron firing and causes
hyperalgesia in diabetic neuropathy. Nature Medicine, 18,
926-933. doi:10.1038/nm.2750
[10] Bierhaus, A., Haslbeck, K.M., Humpert, P.M., Liliensiek,
B., Dehmer, T., Morcos, M., et al. (2004) Loss of pain
perception in diabetes is dependent on a receptor of the
immunoglobulin superfamily. Journal Clinical Investiga-
tion, 114, 1741-1751.
[11] Cameron, N.E., Gibson, T.M., Nangle, M.R. and Cotter,
M.A. (2005) Inhibitors of advanced glycation end product
formation and neurovascular dysfunction in experimental
diabetes. Annuals New York Academy Science, 1043, 784-
792. doi:10.1196/annals.1333.091
[12] Nagamatsu, M., Nickander, K.K., Schmelzer, J.D., Raya,
A., Wittrock, D.A., Tritschler, H., et al. (1995) Lipoic
acid improves nerve blood flow, reduces oxidative stress,
and improves distal nerve conduction in experimental
diabetic neuropathy. Diabetes Care, 18, 1160-1167.
doi:10.2337/diacare.18.8.1160
[13] Cameron, N.E., Tuck, Z., McCabe, L. and Cotter, M.A.
(2001) Effect of the hydroxyl radical scavenger, dime-
thylthiourea, on peripheral nerve tissue perfusion, con-
duction velocity and nociception in experimental diabetes.
Diabetologia, 44, 1161-1169.
doi:10.1007/s001250100626
[14] Coppey, L.J., Gellett, J.S., Davidson, E.P., Dunlap, J.A.,
Lund, D.D. and Yorek, M.A. (2001) Effect of antioxidant
treatment of streptozotocin-induced diabetic rats on en-
doneurial blood flow, motor nerve conduction velocity,

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110 109
and vascular reactivity of epineurial arterioles of the sci-
atic nerve. Diabetes, 50, 1927-1937.
doi:10.2337/diabetes.50.8.1927
[15] Obrosova, I.G., Mabley, J.G., Zsengellér, Z., Charni-
auskaya, T., Abatan, O.I., Groves, J.T., et al. (2005) Role
for nitrosative stress in diabetic neuropathy: Evidence
from studies with a peroxynitrite decomposition catalyst.
FASEB Journal, 19, 401-403.
[16] Lupachyk, S., Shevalye, H., Maksimchyk, Y., Drel, V.R.
and Obrosova, I.G. (2011) PARP inhibition alleviates dia-
betes-induced systemic oxidative stress and neural tissue
4-hydroxynonenal adduct accumulation: Correlation with
peripheral nerve function. Free Radical Biology Medicine,
50, 1400-1409. doi:10.1016/j.freeradbiomed.2011.01.037
[17] Goss, J.R., Goins, W.F., Lacomis, D., Mata, M., Glorioso,
J.C. and Fink, D.J. (2002) Herpes simplex-mediated gene
transfer of nerve growth factor protects against peripheral
neuropathy in streptozotocin-induced diabetes in the
mouse. Diabetes, 51, 2227-2232.
doi:10.2337/diabetes.51.7.2227
[18] Bianchi, R., Buyukakilli, B., Brines, M., Savino, C.,
Cavaletti, G., Oggioni, N., et al. (2004) Erythropoietin
both protects from and reverses experimental diabetic
neuropathy. Proceedings National Academy Science, 101,
823-828. doi:10.1073/pnas.0307823100
[19] Nakamura, J., Kato, K., Hamada, Y., Nakayama, M., Chaya,
S., Nakashima, E., et al. (1999) A protein kinase C-beta-
selective inhibitor ameliorates neural dysfunction in strep-
tozotocin-induced diabetic rats. Diabetes, 48, 2090-2095.
doi:10.2337/diabetes.48.10.2090
[20] Cameron, N.E., Cotter, M.A., Jack, A.M., Basso, M.D.
and Hohman, T.C. (1999) Protein kinase C effects on
nerve function, perfusion, Na(+), K(+)-ATPase activity
and glutathione content in diabetic rats. Diabetologia, 42,
1120-1130. doi:10.1007/s001250051280
[21] Li, F., Drel, V.R., Szabó, C., Stevens, M.J. and Obrosova,
I.G. (2005) Low-dose poly(ADP-ribose) polymerase in-
hibitor-containing combination therapies reverse early
peripheral diabetic neuropathy. Diabetes, 54, 1514-1522.
doi:10.2337/diabetes.54.5.1514
[22] Obrosova, I.G., Xu, W., Lyzogubov, V.V., Ilnytska, O.,
Mashtalir, N., Vareniuk, I., et al. (2008) PARP inhibition
or gene deficiency counteracts intraepidermal nerve fiber
loss and neuropathic pain in advanced diabetic neuropa-
thy. Free Radical Biology Medicine, 44, 972-981.
doi:10.1016/j.freeradbiomed.2007.09.013
[23] Kellogg, A.P., Wiggin, T.D., Larkin, D.D., Hayes, J.M.,
Stevens, M.J. and Pop-Busui, R. (2007) Protective effects
of cyclooxygenase-2 gene inactivation against peripheral
nerve dysfunction and intraepidermal nerve fiber loss in
experimental diabetes. Diabetes, 56, 2997-3005.
doi:10.2337/db07-0740
[24] Stavniichuk, R., Drel, V.R., Shevalye, H., Vareniuk, I.,
Stevens, M.J., Nadler, J.L., et al. (2010) Role of 12/15-
lipoxygenase in nitrosative stress and peripheral predia-
betic and diabetic neuropathies. Free Radical Biology
Medicine, 49, 1036-1045.
doi:10.1016/j.freeradbiomed.2010.06.016
[25] Obrosova, I.G., Stavniichuk, R., Drel, V.R., Shevalye, H.,
Vareniuk, I., Nadler, J.L., et al. (2010) Different roles of
12/15-lipoxygenase in diabetic large and small fiber pe-
ripheral and autonomic neuropathies. American Journal
Pathology, 177, 1436-1447.
doi:10.2353/ajpath.2010.100178
[26] Purves, T., Middlemas, A., Agthong, S., Jude, E.B.,
Boulton, A.J., Fernyhough, P., et al. (2001) A role for mi-
togen-activated protein kinases in the etiology of diabetic
neuropathy. FASEB Journal, 15, 2508-2514.
doi:10.1096/fj.01-0253hyp
[27] Price, S.A., Agthong, S., Middlemas, A.B. and Tomlinson,
D.R. (2004) Mitogen-activated protein kinase p38 medi-
ates reduced nerve conduction velocity in experimental
diabetic neuropathy: Interactions with aldose reductase.
Diabetes, 53, 1851-1856. doi:10.2337/diabetes.53.7.1851
[28] Cheng, H.T., Dauch, J.R., Oh, S.S., Hayes, J.M., Hong, Y.
and Feldman, E.L. (2010) p38 mediates mechanical allo-
dynia in a mouse model of type 2 diabetes. Molecular
Pain, 19, 6-28.
[29] Stavniichuk, R., Drel, V.R., Shevalye, H., Maksimchyk,
Y., Kuchmerovska, T.M., Nadler, J.L., et al. (2011) Bai-
calein alleviates diabetic peripheral neuropathy through
inhibition of oxidative-nitrosative stress and p38 MAPK
activation. Experimental Neurology, 230, 106-113.
doi:10.1016/j.expneurol.2011.04.002
[30] Drel, V.R., Pacher, P., Stavniichuk, R., Xu, W., Zhang, J.,
Kuchmerovska, T.M., et al. (2011) Poly(ADP-ribose)po-
lymerase inhibition counteracts renal hypertrophy and
multiple manifestations of peripheral neuropathy in dia-
betic Akita mice. International Journal Molecular Medi-
cine, 28, 629-635.
[31] Stavniichuk, R., Shevalye, H., Hirooka, H., Nadler, J.L.
and Obrosova, I.G. (2012) Interplay of sorbitol pathway
of glucose metabolism, 12/15-lipoxygenase, and mito-
gen-activated protein kinases in the pathogenesis of dia-
betic peripheral neuropathy. Biochemical Pharmacology,
83, 932-940. doi:10.1016/j.bcp.2012.01.015
[32] Askwith, T., Zeng, W., Eggo, M.C. and Stevens, M.J.
(2012) Taurine reduces nitrosative stress and nitric oxide
synthase expression in high glucose-exposed human
Schwann cells. Experimental Neurology, 233, 154-162.
doi:10.1016/j.expneurol.2011.09.010
[33] Roberts, P.J. and Der, C.J. (2007) Targeting the Raf-
MEK-ERK mitogen-activated protein kinase cascade for
the treatment of cancer. Oncogene, 26, 3291-3310.
doi:10.1038/sj.onc.1210422
[34] Lehmann, H.C. and Höke, A. (2010) Schwann cells as a
therapeutic target for peripheral neuropathies. CNS Neu-
rology Disorders Drug Targets, 9, 801-816.
doi:10.2174/187152710793237412
[35] Lehmann, H.C., Chen, W., Mi, R., Wang, S., Liu, Y., Rao,
M., et al. (2012) Human Schwann cells retain essential
phenotype characteristics after immortalization. Stem
Cells Development, 21, 423-431.
doi:10.1089/scd.2010.0513
[36] Obrosova, I.G., Drel, V.R., Pacher, P., Ilnytska, O., Wang,
Z.Q., Stevens, M.J., et al. (2005) Oxidative-nitrosative
stress and poly(ADP-ribose) polymerase (PARP) activi-
tion in experimental diabetic neuropathy: The relation is
revisited. Diabetes, 54, 3435-3441.

R. Stavniichuk et al. / Journal of Diabetes Mellitus 3 (2013) 101-110
110
doi:10.2337/diabetes.54.12.3435
[37] Stevens, M.J., Li, F., Drel, V.R., Abatan, O.I., Kim, H.,
Burnett, D., et al. (2007) Nicotinamide reverses neuro-
logical and neurovascular deficits in streptozotocin dia-
betic rats. The Journal of Pharmacology and Experimen-
tal Therapeutics, 320, 458-464.
doi:10.1124/jpet.106.109702
[38] Askwith, T., Zeng, W., Eggo, M.C. and Stevens, M.J.
(2009) Oxidative stress and dysregulation of the taurine
transporter in high-glucose-exposed human Schwann
cells: Implications for pathogenesis of diabetic neuropa-
thy. American Journal of Physiology Endocrinology and
Metabolism, 297, 620-628.
doi:10.1152/ajpendo.00287.2009
[39] Pop-Busui, R., Marinescu, V., Van Huysen, C., Li, F.,
Sullivan, K., Greene, D.A., et al. (2002) Dissection of
metabolic, vascular, and nerve conduction interrelation-
ships in experimental diabetic neuropathy by cyclooxy-
genase inhibition and acetyl-L-carnitine administration.
Diabetes, 51, 2619-2628. doi:10.2337/diabetes.51.8.2619
[40] Kellogg, A.P., Converso, K., Wiggin, T., Stevens, M. and
Pop-Busui, R. (2009) Effects of cyclooxygenase-2 gene
inactivation on cardiac autonomic and left ventricular
function in experimental diabetes. American Journal of
Physiology Heart and Circulation Physiology, 296, 453-
461. doi:10.1152/ajpheart.00678.2008
[41] Reddy, M.A., Thimmalapura, P.R., Lanting, L., Nadler,
J.L., Fatima, S. and Natarajan, R. (2002) The oxidized
lipid and lipoxygenase product 12(S)-hydroxyeicosa-tet-
raenoic acid induces hypertrophy and fibronectin tran-
scription in vascular smooth muscle cells via p38 MAPK
and cAMP response element-binding protein activation.
Mediation of angiotensin II effects. Journal of Biological
Chemistry, 277, 9920-9928.
doi:10.1074/jbc.M111305200
[42] Dwarakanath, R.S., Sahar, S., Reddy, M.A., Castanotto,
D., Rossi, J.J. and Natarajan, R. (2004) Regulation of mono-
cyte chemoattractant protein-1 by the oxidized lipid,
13-hydroperoxyoctadecadienoic acid, in vascular smooth
muscle cells via nuclear factor-kappa B (NF-kappa B).
Journal of Molecular and Cell Cardiology, 36, 585-595.
doi:10.1016/j.yjmcc.2004.02.007
[43] Reilly, K.B., Srinivasan, S., Hatley, M.E., Patricia, M.K.,
Lannigan, J., Bolick, D.T., et al. (2004) 12/15-Lipoxy-
genase activity mediates inflammatory monocyte/endo-
thelial interactions and atherosclerosis in vivo. The Jour-
nal of Biological Chemistry, 279, 9440-9450.
doi:10.1074/jbc.M303857200
[44] Dwarakanath, R.S., Sahar, S., Lanting, L., Wang, N.,
Stemerman, M.B., Natarajan, R., et al. (2008) Viral vec-
tor-mediated 12/15-lipoxygenase overexpression in vas-
cular smooth muscle cells enhances inflammatory gene
expression and migration. Journal of Vascular Research,
45, 132-142. doi:10.1159/000109966
[45] Reddy, M.A., Sahar, S., Villeneuve, L.M., Lanting, L. and
Natarajan, R. (2009) Role of src tyrosine kinase in the
atherogenic effects of the 12/15-lipoxygenase pathway in
vascular smooth muscle cells. Arteriosclerosis, Thrombo-
sis and Vascular Biology, 29, 387-393.
doi:10.1161/ATVBAHA.108.179150
[46] Kim, Y.S., Reddy, M.A., Lanting, L., Adler, S.G. and
Natarajan, R. (2003) Differential behavior of mesangial
cells derived from 12/15-lipoxygenase knockout mice
relative to control mice. Kidney International, 64, 1702-
1714. doi:10.1046/j.1523-1755.2003.00286.x
[47] Nangle, M.R., Cotter, M.A. and Cameron, N.E. (2006)
Correction of nitrergic neurovascular dysfunction in dia-
betic mouse corpus cavernosum by p38 mitogen-activated
protein kinase inhibition. International Journal of Impo-
tence Research, 18, 258-263. doi:10.1038/sj.ijir.3901414
[48] Daulhac, L., Mallet, C., Courteix, C., Etienne, M., Dur-
oux, E., Privat, A.M., et al. (2006) Diabetes-induced me-
chanical hyperalgesia involves spinal mitogen-activated
protein kinase activation in neurons and microglia via
N-methyl-D-aspartate-dependent mechanisms. Molecular
Pharmacology, 70, 1246-1254.
doi:10.1124/mol.106.025478
[49] Du, Y., Tang, J., Li, G., Berti-Mattera, L., Lee, C.A.,
Bartkowski, D., et al. (2010) Effects of p38 MAPK inhi-
bition on early stages of diabetic retinopathy and sensory
nerve function. Investigative Ophthalmology & Visual
Science, 51, 2158-2164. doi:10.1167/iovs.09-3674
[50] Daulhac, L., Maffre, V., Mallet, C., Etienne, M., Privat,
A.M., Kowalski-Chauvel, A., et al. (2011) Phosphoryla-
tion of spinal N-methyl-d-aspartate receptor NR1 sub-
units by extracellular signal-regulated kinase in dorsal
horn neurons and microglia contributes to diabetes-in-
duced painful neuropathy. European Journal of Pain, 15,
169.e1-169.e12.
[51] Tsuda, M., Ueno, H., Kataoka, A., Tozaki-Saitoh, H. and
Inoue, K. (2008) Activation of dorsal horn microglia con-
tributes to diabetes-induced tactile allodynia via extracel-
lular signal-regulated protein kinase signaling. Glia, 56,
378-386. doi:10.1002/glia.20623
[52] Price, S.A., Gardiner, N.J., Duran-Jimenez, B., Zeef, L.A.,
Obrosova, I.G. and Tomlinson, D.R. (2006) Thioredoxin
interacting protein is increased in sensory neurons in ex-
perimental diabetes. Brain Research, 1116, 206-214.
doi:10.1016/j.brainres.2006.07.109
[53] Bürger, F., Krieg, P., Marks, F. and Fürstenberger, G.
(2000) Positional- and stereo-selectivity of fatty acid oxy-
genation catalysed by mouse (12S)-lipoxygenase iso-en-
zymes. Biochemical Journal, 348, 329-335.
doi:10.1042/0264-6021:3480329
[54] Gong, Y.Z., Ding, W.G., Wu, J., Tsuji, K., Horie, M. and
Matsuura, H. (2008) Cinnamyl-3,4-dihydroxy-alpha cya-
nocinnamate and nordihydroguaiaretic acid inhibit human
Kv1.5 currents independently of lipoxygenase. European
Journal of Pharmacology, 600, 18-25.
doi:10.1016/j.ejphar.2008.10.010
[55] Pergola, C., Jazzar, B., Rossi, A., Buehring, U., Luderer,
S., Dehm, F., et al. (2011) Cinnamyl-3,4-dihydroxy-α-
cyanocinnamate is a potent inhibitor of 5-lipoxygenase.
Journal of Pharmacology and Experimental Therapeutics,
338, 205-213. doi:10.1124/jpet.111.180794
[56] Wen, Y., Scott, S., Liu, Y., Gonzales, N. and Nadler, J.L.
(1997) Evidence that angiotensin II and lipoxygenase
products activate c-Jun NH2-terminal kinase. Circulation
Research, 81, 651-655.doi:10.1161/01.RES.81.5.651