Selenoproteins and Selenium-dependent Redox
Signaling Alter Diabetes Risk
Dmitri Fomenko
Department of Biochemistry and Redox
Biology Center
University of Nebraska-Lincoln
Selenium is essential for human health
Selenium deficiency may cause
• Increased risk of cancer
• Type 2 diabetes
• Increased risk of AIDS in HIV-infected
individuals
• Male reproduction problems
• Increased risk of viral infection
• Keshan, Kashin-Beck diseases
Selenium overnutrition may cause type 2
diabetes
Selenium – link to cancer and diabetes
Se has been shown to be effective in reducing the incidence of cancer in animal
models and human clinical trials. Of the more than 100 reported studies that have
examined the chemopreventive potential of Se, approximately two-thirds
demonstrated a protective effect, and the remaining studies reported no effect.
A interesting and unexpected side effect of Se supplementation was demonstrated
for Nutritional Prevention of Cancer (NPC) and SELECT trials.
NPC showed about a 50% increase in incidence of type 2 diabetes in the Sesupplemented group, which was associated with the high initial Se levels in
plasma.
The SELECT trial demonstrated high incidence of type 2 diabetes in men taking
Se supplements. This observation was used as one of the reasons for trial
termination.
Taken together, these observations suggest that Se supplementation of people
with high initial plasma Se may significantly increase the risk of diabetes
development. A subsequent study showed that both, high and low Se
supplementation is associated with hyperinsulinemia and decreased insulin
sensitivity. The molecular mechanisms for the effect of dietary Se on the
development of type 2 diabetes are not understood.
• Se is present in proteins in the form of 21st
amino acid – selenocysteine. More than 90% of
the Se pool in mammals is exist in
selenocysteine form
• Selenocysteine is encoded by UGA codon,
however, UGA is also a stop signal
• mRNA structure, selenocysteine insertion
sequence (SECIS) element, is essential for
insertion of selenocysteine
Selenocysteine insertion in eukaryotes
Sec
EFsec
SBP2
AUG
UGA
UAA/UAG
Selenocysteine versus Cysteine
Selenocysteine
Cysteine
UGA codon
pKa = 5.2
UGU, UGC-codons
pKa = 8.3
Se- S
S- S
S
S
Sec serves redox function!
Selenoproteomes
Eukaryotes:
0-57
(Aureococcus)
Bacteria:
0-57
(worm symbiont)
Archaea:
0-9
(M. maripaludis)
Viruses:
0-1
(fowlpox virus)
Model organisms
Mouse, rat
Zebrafish
Drosophila
C. elegans
S. cerevisiae, Arabidopsis
Selenoproteins
24
37
3
1
0
Human Selenoproteome – 25 selenoproteins
Selenoprotein
GPX1
GPX2
GPX3
GPX4
GPX6
SelP
DI1
DI2
DI3
SelW
SelV
SelT
SelH
SelM
Sep15
TR1
TR2
TR3
SelS
SelK
SelO
SelI
SelN
SelR
SPS2
Sec-motif
UxxT
UxxT
UxxT
UxxT
UxxT
UxxC
SxxU
SxxU
SxxU
CxxU
CxxU
CxxU
CxxU
CxxU
CxU
U
U
U
U
U
U
U
U
UxxS
UxC
Protein secondary structure
Selenoprotein expression and selenium supplementation
75Se
metabolic labeling
in HEK293 cells
Stress response selenoproteins
Gpx1, SelR, SelT, SelW
kDa
Housekeeping selenoproteins
TR1, TR2, TR3, GPX4,
TR1
Stress response selenoprotein expression is
regulated by dietary selenium
GPX1
GPX4
Sep15
SelW
Novoselov et al, ARS, 2009
Available information regarding selenoproteins and diabetes
1. Glutathione peroxidase 1 (GPx1)
Transgenic mice overexpressing Sec containing GPx1 develop obesity and
insulin resistance phenotype.
GPx1 knockout show low insulin level and high insulin sensitivity, however,
positive effect of knockout was diminished by antioxidant supplementation.
Gpx1 is a major hydroperoxides scavenger in mammalian cells. This
selenoprotein account more than 90% of GPx activity in the cell and strongly
regulated with Se. Thus, high Se is associated with high GPx1 activity,
disrupted H2O2 signaling, and redox stress.
2. Selenoprotein S (SelS)
SelS association with type 2 diabetes in an animal model was demonstrated.
SelS is down-regulated in liver, adipose tissue, and skeletal muscle in the fed
state of diabetic animal models in comparison with healthy animals. SelS was
found to be insulin regulated and its expression was increased after insulin
stimulation of human adipocytes.
The goal of this project is to investigate the roles of Se and selenoproteins
regulation of glucose homeostasis
Considering Se function in the context of thiol oxidoreductases, we suggests that
redox processes play a critical role in glucose homeostasis.
We suggest that insulin signaling pathways are crosslinked with hydrogen
peroxide-mediated signaling and selenoproteins, known as a subgroup of thiol
oxidoreductases and contribute to the insulin signaling pathway by controlling the
hydrogen peroxide concentration and redox homeostasis in the cell.
Proposed project will be the first attempt to explain regulation of glucose
homeostasis by Se, and the first attempt to explain mechanisms of the redox
control of insulin signaling by selenoproteins.
CENTRAL HYPOTESIS
High dietary Se
Low dietary Se
Low glutathione peroxidases activity
Oxidative stress, high H2O2
High glutathione peroxidases activity
Redox stress, disrupted H2O2 mediated signaling
PTPs, unknown mechanism
unknown mechanism
Hyperinsulinemia and insulin resistance – type 2 diabetes
Overloaded b-cells and ER stress
Unfolded protein response
(Sep15, SelM, SelS, SelK)
Cells adapt - homeostasis
Type 1 diabetes
Specific Aim 1: Clarify the role of Se and selenoproteins in type 2
diabetes development. This aim will be accomplished through completion
of two sub-aims:
1.1 To investigate the role of Se and selenoproteins in glucose
homeostasis. We will utilize several knockout and transgenic models to
demonstrate contribution of different selenoproteins in type 2 diabetes
development.
1.2 To determine mechanistic insides of Se and selenoproteins in insulin
signaling. The roles of Se containing thiol peroxidases in insulin signaling will
be addressed.
1.1 To investigate the role of Se and selenoproteins in glucose homeostasis.
Experimental Design. We will employ transgenic/knockout mouse models, in which
expression of either stress-related selenoproteins or the entire selenoprotein class is affected
in the skeletal muscle of animals receiving a normal supply of dietary Se. In addition, we will
estimates glucose homeostasis in GPx1 KO mouse.
Type of animals
Genotype
Conditional knockout (skeletal
muscle myocites)
ΔSec tRNA
Transgenic
IA
Knockout
ΔGPx1
6
Description
No Sec tRNA expression in skeletal muscle myocites , and
consequently no selenoprotein expression in this cell type,
selenoprotein expression in other cell types is not affected
Overexpression of Sec tRNA transgene, low levels of stress-related
selenoproteins (e.g., GPx1); expression of essential selenoproteins
is not affected
No GPx1 expression, increased insulin sensitivity, low level of
insulin and resistance to high fat diet
Skeletal muscle conditional Sec tRNA KO model will be generated using floxed Sec
tRNA transgenic mouse.
Groups of seven male animals of this model with control will be subjected to six different
diets (Harland TekLad) containing 0 parts per million Se (Se-deficient), 0.1ppm Se and
0.4ppm Se, and a similar custom made diet based on a high fat diet TD.06414. The
same groups of animals will be subjected to the same diets with additional
supplementation with 20mM NAC in water.
Glucose homeostasis parameters. After one month of dietary supplementation we will
start weekly measurement of fasting or fed state plasma glucose, Se, and insulin levels.
Glucose tolerance tests will be performed in 12 hour fasted animals by injection of
glucose followed by plasma glucose measurements every 60 minutes. The Se level in the
plasma will be estimated using the ICPMS. Insulin sensitivity tests will be performed on
12 hour fasted animals by intraperitoneal injection of insulin. Plasma glucose will be
measured every 60 minutes. Selenoprotein expression will be estimated by Western Blot
and 75Se metabolic labeling. All animals will be euthanized 18 weeks after beginning the
experiment and tissues will be collected for redox parameters measurement.
Similar experiment will be performed with I6A and GPx1 KO animal model.
All collected samples will be analyzed for redox stress parameters.
Expected Outcomes of Specific Aim 1.1. We expect to observe type 2 diabetes-like
phenotype for both the Sec tRNA KO and transgenic models at all Se concentrations. We
expect this phenotype will be more pronounced for a high fat diet. NAC supplementation
will partially compensate Se deficiency in KO models according to our central hypothesis.
Control groups will show type 2 diabetes-like phenotype for both a low and high Se diet.
This experiment will help us determine the contribution of housekeeping and stress
response selenoproteins in glucose homeostasis. The experiment on the GPx1 knockout
model will allow us to identify additional selenoproteins that are involved in diabetic
phenotype development.
1.2 To Determine mechanistic insides of Se and selenoproteins in insulin signaling.
We demonstrated the roles of thiol peroxidases in H2O2 mediated signaling using yeast system.
Thiol peroxidases oxidize regulatory and signaling proteins, resulting in transcriptional response
and signaling programs (Fomenko et al, PNAS, 2011).
Thioredoxin
reductases
Peroxiredoxins
Thioredoxins
Kinases
NADPH
Signaling
Transcription
factors
H2O2
Other
targets
Glutathione
reductase
Glutaredoxins
Glutathione
peroxidases
Glutathione
Protein tyrosine phosphatases, including PTP1, could be reversibly inactivated by H2O2
oxidation. PTP1 is responsible for control of phosphorylation status of the insulin receptor.
Insulin stimulation of mammalian cells is associated with Nox4-mediated H2O2 production, which
causes reversible oxidization and inhibition of PTP1B activity and promotes insulin signaling.
Recombinant PTP1 oxidation was demonstrated at 75mM of H2O2 and higher; however,
mammalian cells induce apoptosis at such concentrations. Considering our observation for yeast
cells, we hypothesize that PTP1 oxidation is mediated by thiol peroxidases – GPxs and/or PRxs
and may be regulated by Se. We suggest that mammalian GPxs, other than GPx1, and PRxs
mediate PTP1 oxidation in insulin signaling pathways.
Experiment Design.
1. The HEK293 cell line is established model system and will be used for insulin signaling
studies. We will use Dharmacon siGENOME SMARTpool siRNAs to knockdown GPxs
and PRxs. The cells will be transfected with siRNAs and treated with 10mM H2O2 after two
days. Phosphorylation status of insulin receptor b-subunit will be estimated by Western
Blot with phospho-specific antibodies.
2. In addition, we will estimate efficiency of oxidation of recombinant PTP1 protein with H2O2
and oxidized recombinant GPx3 from yeast.
3.
Phosphorylation status of insulin receptor b subunit will be estimated for miocytes of
animal models from Sub-aim 1.1
Expected Outcomes of Specific Aim 1.2 We will determine the contribution of Sec containing
GPxs in insulin receptor regulation through reversible oxidation of protein tyrosine
phosphatases. We expect that thiol peroxidases, other than GPx1, are involved in the
transferring of oxidative equivalent from H2O2 to PTP1. This process may be regulated by Se
supplementation.
Specific Aim 2: Characterize the role of Se and selenoproteins in the
protection of pancreatic b-cells from ER stress and oxidative stress. This
aim will be accomplished through completion of two sub-aims:
2.1 Determine the role of Se and selenoproteins in ER stress defense. We
expect to determine how Se supplementation influences ER stress response and
protects pancreatic b-cells from ER stress-mediated apoptosis.
2.2 Investigate the protective role Se and selenoproteins play in oxidative
protein folding induced oxidative stress. The contribution of different
selenoproteins in protection from protein folding associated oxidative stress in
pancreatic b-cells will be addressed.
2.1 To determine the roles of Se and selenoproteins in ER stress defense.
ER is characterized by very intense and diverged redox processes that are catalyzed by
numerous thiol oxidoreductases and selenoproteins. Seven of the 25 identified human
selenoproteins are localized in ER.
Hyperinsulinemia is associated with increased insulin synthesis and secretion. Pancreatic bcells overload, resulting in ER stress and activation of the ER stress-mediated apoptosis
pathway. b-cells death is one of the central features in conversion of type 2 diabetes to type
1. ER selenoproteins are regulated by Se supplementation and serve in protein folding and
ER stress related pathways. Thus, Se may contribute in pancreatic b-cells protection from ER
stress and delay or prevent apoptosis.
Translocon
Export
Secretory pathway
Protein
glycosylation
Oxidative folding
OST
SelT, SelN, DI2
CYTOSOL
Glycoprotein folding
quality control
Chaperones
ER
UGT, Sep15
SelM
Retrotranslocation SelS, SelK
Cytosolic degradation
Experiment Design. b-cells conditional Sec tRNA KO model will be generated using floxed Sec
tRNA transgenic mouse.
Groups of seven male animals of this model and seven male animals of control groups will be
subjected to the high fat diet TD.06414 (Harland TekLad) to initiate type 2 diabetes.
After five month of dietary supplementation we will begin monthly measurement of glucose
homeostasis parameters as described in Specific Aim 1.1 for control of diabetes progression.
All animals will be euthanized after one year, pancreatic tissue will be collected, and
expression of each ER selenoprotein will be analyzed with Western Blot using specific
antibodies available in project leader’s laboratory.
ER stress will be verified by the expression level of the BiP chaperone in all collected tissues
using Western blot. The UPR will be analyzed by monitoring the phosphorylation status of
eukaryotic initiation factor eIF2α. The EOR will be examined by monitoring the phosphorylation
status of NF-κB. Similar analysis of control groups from Specific Aim 1.1.
Expected Outcomes of Specific Aim 2.1 This experiment will help to determine contribution of
Se and ER selenoproteins in ER stress protection.
2.2 Investigate the protective role Se and selenoproteins play in oxidative
protein folding induced oxidative stress.
Oxidative folding in the ER is associated with formation of H2O2 molecules for each formed
disulfide bond. The insulin molecule contains three disulfides and its increased synthesis may
be linked with strong oxidative stress with subsequent b-cells death.
At the same time, b-cells have only 1% of catalase, 2% of GPx, and 29% of SOD activity
compared to liver cells. In this experiment, we will estimate the roles of different
selenoproteins in b-cells protection from oxidative stress associated death.
Experiment Design. I6A transgenic model and corresponding control samples of b-cells from
Specific Aim 1.1, and b-cells conditional Sec tRNA KO model samples will be analyzed for
oxidative damage. Selenoprotein expression will be estimated by Western Blot and 75Se
metabolic labeling. This experiment will allow us to address the contribution of housekeeping
and stress related selenoproteins in b-cells protection from oxidative stress.
Expected Outcomes of Specific Aim 2.2. We expect to observe a protective role of Se and ER
selenoproteins in ER stress conditions. In addition, we expect that Se and selenoproteins are
involved in b-cells protection from protein folding-mediated oxidative stress.
Summary of expected outcome:
1. We will determine roles of Se and particular selenoproteins in glucose
homeostasis control.
2. We will show the roles of Sec-containing glutathione peroxidases in
insulin signaling pathways.
3. We will determine the roles of Se and selenoproteins in protection of
pancreatic b-cells from ER stress and oxidative stress.
Proposed project and NPOD mission
Selenium is essential micronutrient in human diet. Our study will help to explore
mechanisms of Se associated diabetes development and will help to redefine an
optimal level of dietary Se, as well as the upper subtoxic limit, to provide health
benefits in cancer prevention without risk of diabetes development.
Current research projects and link to nutrigenomics:
This project is logically based on my recent work. I have strong experience in
selenium biology and more than 20 of my recent publications are related to
identification and functional characterization of selenoproteins. I developed and
characterized several selenoprotein knockout mouse models.
One of my side-project is related to the boron metabolism and identification of
the mechanisms of boron toxicity.