In the present study we investigated whether PHB is expressed in b-cells and protects these cells against deleterious effects of ethanol, using INS-1E and RINm5F b-cell lines.. Thus, PHB
Trang 1Prohibitin is expressed in pancreatic b-cells and protects against oxidative and proapoptotic effects of ethanol
Jong Han Lee1, K Hoa Nguyen1, Suresh Mishra1,2and B L Gre´goire Nyomba1,2
1 Department of Physiology, Diabetes Research Group, University of Manitoba, Winnipeg, Canada
2 Department of Internal Medicine, Diabetes Research Group, University of Manitoba, Winnipeg, Canada
Introduction
Pancreatic b-cell dysfunction is a prerequisite for the
development of type 2 diabetes The prevalence of type
2 diabetes is related to lifestyle choices, such as high
calorie diets, lack of physical activity and smoking
Alcoholism is a known risk factor for type 2 diabetes,
although moderate ethanol consumption may have
health benefits [1] The diabetogenic effects of ethanol may include its contribution to excess caloric intake and obesity, induction of pancreatitis and impairment
of liver function [2] Recent studies have found that ethanol increases insulin resistance in liver and skeletal muscle [3–5] However, a limited number of studies
Keywords
apoptosis; oxidative stress; prohibitin; b-cells
Correspondence
B L G Nyomba, Diabetes Research Group,
University of Manitoba, 715 McDermot
Avenue Room 834, Winnipeg, Manitoba,
Canada R3E 3P4
Fax: +1 204 789 3940
Tel: +1 204 789 3697
E-mail: bnyomba@cc.umanitoba.ca
(Received 19 October 2009, revised 12
November 2009, accepted 19 November
2009)
doi:10.1111/j.1742-4658.2009.07505.x
Pancreatic b-cell dysfunction is a prerequisite for the development of type 2 diabetes Alcoholism is a diabetes risk factor and ethanol increases oxida-tive stress in b-cells, whereas the mitochondrial chaperone prohibitin (PHB) has antioxidant effects in several cell types In the present study we investigated whether PHB is expressed in b-cells and protects these cells against deleterious effects of ethanol, using INS-1E and RINm5F b-cell lines Endogenous PHB was detected by western blot and immunocyto-chemistry Reactive oxygen species were determined by 5-(and-6)-chloro-methyl-2¢,7¢-dichlorodihydrofluorescein diacetate fluorescence assay, and mitochondrial activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, uncoupling protein 2 expression and ATP production Cell death was determined by Hoechst
33342 staining, cleaved caspase-3 levels and flow cytometry PHB was expressed in b-cells under normal conditions and colocalized with Hoechst
33342 in the nucleus and with the mitochondrial probe Mitofluor in the perinuclear area In ethanol-treated cells, MTT reduction and ATP produc-tion decreased, whereas reactive oxygen species, uncoupling protein 2 and cleaved caspase-3 levels increased In addition, flow cytometry analysis showed an increase of apoptotic cells Ethanol treatment increased PHB expression and induced PHB translocation from the nucleus to the mito-chondria PHB overexpression decreased the apoptotic effects of ethanol, whereas PHB knockdown enhanced these effects The protective effects of endogenous PHB were recapitulated by incubation of the cells with recombinant human PHB Thus, PHB is expressed in b-cells, increases with oxidative stress and protects the cells against deleterious effects of ethanol
Abbreviations
CM-H2DCF, 5-(and-6)-chloromethyl-2¢,7¢-dichlorodihydrofluorescein; CM-H 2 DCFDA, 5-(and-6)-chloromethyl-2¢,7¢-dichlorodihydrofluorescein diacetate; FITC, fluorescein isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NaCl ⁄ P i , phosphate-buffered saline; PHB, prohibitin; ROS, reactive oxygen species; SEM, standard error of the mean; siRNA, short inhibitory RNA; UCP2, uncoupling protein 2.
Trang 2have reported on deleterious effects of ethanol on
b-cells, where ethanol inhibited insulin secretion [6–8]
Excessive ethanol consumption leads to cell injury
through the production of reactive oxygen species
(ROS) and mitochondrial dysfunction [9,10] Increased
ROS production is one of the earliest events in glucose
intolerance and it may be a mechanism of pancreatic
b-cell dysfunction in type 2 diabetes, as b-cells are very
sensitive to oxidative stress due to their insufficient
antioxidant mechanisms
Prohibitin (PHB), a 30 kDa evolutionarily conserved
protein, is present in multiple cellular compartments
[11], including the cell nucleus [12], the plasma
mem-brane [13–15] and lipid droplets shed from adipocytes
[16] and breast cancer cells [17] PHB is an
anti-inflam-matory [18] and tumor suppressor protein, with
muta-tions occurring in various cancers [19] Recent studies
suggest that PHB may be a regulator of transcription, a
chaperone in the mitochondria [20,21] and a secreted
protein [22] found in the circulation [23] It is possible
that PHB, in its role as a mitochondrial chaperone,
pro-tects cells against oxidative stress [21,24,25] However, it
is not known if PHB is expressed or plays a role in
pan-creatic b-cells The objective of the present study was to
investigate the expression and antioxidant effects of
PHB in pancreatic b-cells exposed to ethanol
Results
Effects of ethanol and recombinant PHB on b-cell
mitochondrial function and apoptosis
We first generated a dose–response curve of ethanol
toxicity using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay in INS-1E
cells incubated with ethanol for 24–48 h (Fig 1) At
24 h there was a statistically significant deleterious
effect starting at 80 mm ethanol (Fig 1A), confirming
our previous report in RINm5F cells [26] At this
con-centration, MTT reduction was decreased by 30%
(Fig 1A); this effect of ethanol did not increase further
after treatment of INS-1E cells for 48 h (Fig 1B) This
effect was comparable with that observed in RINm5F
cells at a low glucose concentration of 5.5 mm
(Fig 2A), which is much lower than glucose
concen-trations recommended for b-cell culture [27] We then
determined ROS production in RINm5F cells and
found that ethanol increased ROS production by 43%,
unlike the low glucose concentration, which decreased
ROS production by 80% compared with the high
glucose concentration (Fig 2B)
Because oxidative stress can induce uncoupling
pro-tein 2 (UCP2) expression at the expense of ATP
synthe-sis [28], we then determined the level of UCP2 protein and ATP production in RINm5F cells Ethanol increased the UCP2 protein level by 42% (Fig 2C), in direct proportion with ROS production, whereas ATP production decreased by 40% (Fig 2D), in inverse proportion with the UCP2 protein level
Because exogenously applied PHB has been shown
to regulate cell metabolism in adipocytes [29], we were also interested in the effects of recombinant PHB on b-cells In ethanol-exposed cells, ROS production (Fig 2B) and UCP2 protein levels (Fig 2C) were both significantly reduced, whereas ATP production was increased, by exogenous PHB treatment (Fig 2D)
To explore ethanol toxicity further, we examined b-cell apoptosis using flow cytometry with fluorescein isothiocyanate (FITC)–annexin V staining, the cleaved caspase-3 assay and Hoechst 33342 nuclear staining In cells exposed to ethanol, flow cytometry revealed the apoptotic cell number to be increased by approximately twofold (Fig 3A–F), whereas the cleaved caspase-3 level increased by 40% (Fig 3G) Recombinant PHB pre-vented b-cell apoptosis, as demonstrated by the normal number of apoptotic cells shown by flow cytometry
0 20 40 60 80 100 120
0 20 m M 40 m M 60 m M 80 m M 100 m M
ETOH
0 20 40 60 80 100 120
0 20 m M 40 m M 80 m M 120m M 160 m M 200 m M
ETOH
*
*
A
B
Fig 1 Effect of ethanol on MTT reduction in INS-1E cells INS-1E cells were incubated for 24 (A) or 48 h (B) in RPMI 1640 medium containing various concentrations of ethanol The results are expressed as a percentage of the control (no ethanol) and shown
as the mean ± SEM N = 4 experiments *P < 0.05 versus control.
Trang 3(Fig 3A–F) and a reduction in the cleaved caspase-3
level (Fig 3G) In addition, with Hoechst staining the
nuclei appeared small and condensed after ethanol
expo-sure (Fig 4A), also consistent with increased apoptosis,
but had a normal appearance after PHB treatment
Cellular distribution of exogenous PHB
The fact that exogenous PHB had protective effects
against ethanol prompted us to determine the cellular
distribution of recombinant His-tagged PHB Using a
fluorescence microscope, the His-tagged PHB did not
localize to the nucleus, but showed a perinuclear
distri-bution and colocalized with the mitochondrial dye
MitofluorTM Red 589, confirming translocation of
exogenous PHB to the mitochondria (Fig 4B,C) A
western blot of total cellular protein extracts using
anti-PHB serum showed two bands in cells incubated
with recombinant PHB, the top band corresponding to
His-tagged PHB (Fig 5A) These data indicate that
exogenous PHB enters the cells
PHB is expressed in b-cells and increased by
ethanol
To determine the expression of PHB in b-cells we first
analyzed the PHB protein level by western blot and
mRNA expression We confirmed that PHB protein
was present in b-cells, had a tendency to increase at
the low (5.5 mm) compared with the high (25 mm) glu-cose concentrations, and clearly increased by 92% in cells treated with ethanol (Fig 5A,B) PHB mRNA expression showed a similar expression pattern as the protein level (Fig 5C) To confirm mitochondrial localization, cell extracts were fractionated prior to western blot analysis In cells exposed to ethanol, wes-tern blot analysis showed a decrease in PHB protein in the nuclear fraction (Fig 5D) and an increase in the cytoplasmic fraction (Fig 5E), suggesting PHB protein exclusion from the nucleus, whereas examination of mitochondrial extracts indicated localization of PHB
to the mitochondria (Fig 5F,G) We also used immu-nocytochemistry and found endogenous PHB to be present in the nucleus and in the perinuclear area (Fig 6C), the latter suggesting mitochondrial localiza-tion
Endogenous PHB protects b-cells against ethanol toxicity
Because endogenous PHB has been reported to have antiapoptotic effects in other cell systems [12,30], we sought to investigate whether it protects b-cells against ethanol toxicity PHB overexpression (Fig 7A) decreased the cleaved caspase-3 level (Fig 7B) and increased MTT reduction (Fig 7C) in ethanol-treated cells PHB had a similar effect in cells treated with
H2O2 In other experiments, cells were transfected with
0 20 40 60 80 100 120 140 160 180
P < 0.01 P < 0.05 P = 0.08 P < 0.01
0 200 400 600 800
P < 0.05
P < 0.01
P < 0.01 P < 0.01
P < 0.01 P < 0.01
P < 0.01
P < 0.05
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
UCP2
Actin
P < 0.01 P < 0.01
0
20
40
60
80
100
120
Fig 2 Effect of ethanol and PHB in RINm5F cells RINm5F cells were incubated for 24 h with or without ethanol (E, 80 mM) and with or without PHB (P, 10 nM) in the presence of glucose (G1: 5.5 mM, G2:
25 mM) MTT (A; n = 3–7 experiments), ROS level (B; n = 3 experiments); UCP2 protein level (C; n = 3 experiments) and ATP production (D; n = 4 experiments) were determined as described in Materials and methods The results are expressed as the mean ± SEM percentage of G2 for (A) and (B); as the mean ± SEM arbitrary units rela-tive to actin for (C); and as the mean ± SEM for (D).
Trang 4PHB short inhibitory RNA (siRNA) (Fig 8A,B) prior
to ethanol treatment, and this caused an increase in
cleaved caspase-3 in both RINm5F (Fig 8C) and
INS-E1 cells (Fig 8D) In a different approach,
apop-tosis of RINm5F cells was determined by counting free
floating cells (Fig 8E) and found to be enhanced in
ethanol-treated cells transfected with PHB siRNA
Discussion
Here we report for the first time that PHB is expressed
in pancreatic b-cells and may protect these cells against
oxidative stress and apoptosis We induced oxidative
stress and apoptosis with ethanol, which has been
demonstrated in other cell types to cause such
deleteri-ous effects [31,32]
Ethanol has also been shown in a small number of
studies to alter pancreatic b-cell function Rats
chroni-cally fed ethanol showed reduced b-cell volume [33], and it has been reported that ethanol inhibits basal and glucose-stimulated insulin secretion in rat islets [6,34,35] In a recent report, ethanol inhibited b-cell metabolic activity judged by the MTT assay [34], in agreement with the current study We found that etha-nol resulted in b-cell apoptosis with mitochondrial dys-function shown by decreased MTT metabolism and ATP production, and increased ROS production and UCP2 levels These alterations occurred at a physio-logically relevant ethanol level of 80 mm (368 mgÆdL)1) found in the blood of clinically nonintoxicated alcohol drinkers [36] ROS have been implicated in b-cell dys-function and apoptosis in rodent models of diabetes [37–40], and changes in mitochondrial function, includ-ing increased ROS and UCP2 expression, lower ATP and a decreased ATP⁄ ADP ratio have also been docu-mented in b-cells from patients with type 2 diabetes
P < 0.05
P < 0.01
P < 0.01
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
G
P < 0.05
Cleaved caspase 3 Actin
~ 19 kDa
~ 17 kDa
0 5 10 15 20 25 30 35 40
10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3
10 0 10 1 10 2 10 3
10 0 10 1 10 2 10 3
10 0 10 1 10 2 10 3
10 0 10 1 10 2 10 3
10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3
PI log
PI log
PI log
PI log
PI log
Annexin V-FITC log
Annexin V-FITC log
Annexin V-FITC log
F
3.1%
F 23.7%
80 158
2.5%
10.4%
G 12.4%
F
Fig 3 Effect of ethanol and PHB on apoptosis in RINm5F cells RINm5F cells were incubated for 24 h as described in Fig 2 Apoptosis was then determined by flow cytometry Histograms from representative flow cytometry experiments are shown in (A)–(E): (A) G1; (B) G2; (C) G2E; (D) G2P; (E) G2EP The percentage of apoptotic cells (percentage of FITC–annexin V versus propidium iodide-positive cells) obtained from four independent experiments using each treatment are shown as the mean ± SEM in (F) As an additional proof of apoptosis, cleaved caspase-3 was determined in cell extracts by western blot (G) Representative blot of n = 3 experiments showing 17 and 19 kDa caspase-3 cleavage bands and the levels of 19 kDa cleaved caspase-3 expressed as the mean ± SEM arbitrary units relative to actin.
Trang 5[41] Our recent study [26] and present data indicate
that ethanol causes oxidative stress in b-cells, which
could be deleterious, especially because these cells have
very low expression of antioxidant enzymes and are
particularly sensitive to oxidative stress [42]
RINm5F cell mitochondrial metabolism, as
deter-mined by MTT, at a 5.5 mm glucose concentration
was 35% less than that recorded at a 25 mm glucose
concentration, whereas ROS production at the lower
glucose concentration was 80% less than that found
at the high glucose concentration These results are
consistent with increased oxidative phosphorylation,
which is the source of ROS, at glucose concentrations
significantly greater than 5.5 mm in these cells
Although PHB is known to be expressed in many
tissues, this is the first report of its expression in
pan-creatic b-cells Under normal conditions, PHB is found
in cell nuclei and the perinuclear area corresponding to
mitochondria, as reported in other cells [12,30] In
b-cells treated with ethanol, the PHB protein level
increased and was predominantly found in the
mito-chondria This finding is consistent with reports in
breast cancer cells showing that PHB is exported from
the nucleus upon apoptotic signaling [12,30] The increase in PHB and its export from the nucleus with subsequent mitochondrial localization following etha-nol exposure prompted us to investigate whether PHB affected mitochondrial function in these cells Because PHB is found in the circulation, we hypothesized that
it may enter the cells
His-tagged recombinant human PHB was shown to enter the cells and localize to the mitochondria, espe-cially in ethanol-treated cells In addition, exogenous PHB or overexpression of endogenous PHB pre-vented metabolic alterations caused by ethanol, whereas PHB deletion by siRNA enhanced ethanol toxicity These findings are reminiscent of recent reports in granulosa cells, where overexpression of PHB attenuated the ability of staurosporine and serum withdrawal to induce apoptosis [12,25,30,43] When granulosa cells were transfected with a PHB– green fluorescence protein fusion construct, this fusion protein moved from the cytoplasm into the mitochondria and inhibited apoptosis In a more recent study, Theiss et al [24] reported that in inflammatory bowel diseases, PHB localizes primarily
G1
A
B
C
D
Fig 4 Localization of exogenous PHB in RINm5F cells RINm5F cells were incubated for 24 h as described in Fig 2 (n = 3 experi-ments) (A) Hoechst 33342 (nuclei) staining; (B) Mitofluor Red 589 (mitochondria) ing; (C) anti-His (exogenous PHB)-FITC stain-ing; (D) merge The arrows indicate staining
of His-tagged PHB in (C) or both PHB and Mitofluor in (D).
Trang 6to the mitochondria and that PHB overexpression
decreases ROS accumulation in intestinal epithelial
cells and protects these cells from oxidant-induced
depletion of glutathione
It has been reported that PHB plays a chaperone
role in the stabilization of newly synthesized subunits
of mitochondrial respiratory enzymes [44] PHB is
essential for normal mitochondrial development and
its deficiency in yeasts and Caenorhabditis elegans is
associated with deficient mitochondrial function [44]
and a reduced life span [45,46] Our findings in b-cells,
which are in agreement with findings in yeasts, C
ele-gans, and granulosa and intestinal cells, are in
contra-diction with recent observations in other cell types
Vessal et al [29] reported that PHB, when added to
fibroblasts or adipocytes, is a potent inhibitor of
mito-chondrial function Furthermore, they reported that
PHB inhibits the mitochondrial enzyme pyruvate
car-boxylase, thereby depleting oxaloacetate and inhibiting
anaplerosis and, consequently, oxidative
phosphoryla-tion As a consequence, mitochondrial glucose and
fatty acid oxidation was inhibited [29] Through this
mechanism, PHB would be expected to have deleteri-ous effects on glucose-induced insulin secretion from pancreatic b-cells, which is dependent on glucose oxi-dation and mitochondrial ATP biosynthesis [47] Taken together, these observations suggest cell type differences in PHB action that need to be confirmed in further studies
The present study did not address the mechanism whereby PHB is excluded from the nucleus or internal-ized Rastogi et al [48] recently reported that PHB contains a leucine-rich nuclear export signal that facili-tates its cytoplasmic translocation PHB internalization has not been previously reported and its mechanisms are still unknown, but could involve a lipid raft or caveolin-dependent process, as PHB is present on the cell membrane in lipid rafts [17,49]; in some cells, PHB
is abundant in the caveolin-1-rich fractions [50] Cave-olins and lipid rafts are involved in the internalization
of various molecules [51,52] Vessal et al [29] identified
EH domain 2 as a binding partner for PHB, and both
EH domain 2 and PHB have been identified in lipid droplets released from 3T3L1 cells [16] EH domain
G1 G2 G2E G2P G2EP PHB
Histone H1
PHB Actin G1 G2 G2E G2P G2EP
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
P < 0.05
His-PHB PHB
Actin
G1 G2 G2E G2P G2EP His -PHB
30 kDa
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
P = 0.06
P < 0.05
Heat shock protein 60 PHB
G1 G2 G2E G2P G2EP
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
P < 0.05
P < 0.01
E
F
G
B
C
Fig 5 Effect of ethanol and exogenous PHB on PHB expression and localization of endogenous PHB in RINm5F cells RINm5F cells were incubated for 24 h as described in Fig 2 Cell extracts were immunoblotted with anti-PHB (A) A representative western blot of recombinant human PHB run in parallel with cell extracts The 30 kDa endogenous PHB band is seen below the His-tagged recombinant PHB (exogenous PHB) (B) Endogenous PHB protein (30 kDa) expressed as the mean ± SEM arbitrary units relative to actin (n = 3 experiments) (C) PHB mRNA expressed as the mean ± SEM fold of G2 (n = 3 experiments) (D) A representative western blot of PHB in the nuclear fraction with histone H1 as the nuclear marker (n = 3 experiments) (E) A representative western blot of PHB in the cytoplasmic fraction (n = 3 experi-ments) (F) Endogenous PHB protein in the mitochondrial fraction, expressed as the mean ± SEM arbitrary units relative to the mitochondrial marker heat shock protein 60 (n = 3 experiments) (G) A representative western blot of endogenous PHB protein in the mitochondrial fraction.
Trang 7proteins have been shown to be involved in
endocyto-sis and vesicle recycling [53]
In summary, we found that PHB is expressed in
pancreatic b-cells and increases with oxidative stress
induced by ethanol exposure, possibly to protect
b-cells against oxidative and proapoptotic effects of
this drug If PHB protects against oxidative stress
induced by other b-cell toxins, it could be a target for
diabetes prevention or treatment
Materials and methods
Materials
His-tagged recombinant human PHB was purchased from
AmProx American Proteomics (Carlsbad, CA, USA) For
overexpression of PHB, pCMV6 XL5 vector containing the
human PHB gene was obtained from Origene Technologies
reagents were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA, USA): a rabbit polyclonal anti-PHB
serum (sc-28259), His-probe antibody (sc-10806),
anti-his-tone H1 serum (sc-803), heat shock protein 60 antibody
(sc-13966), anti-rabbit-FITC (sc-2012), mouse PHB siRNA
(sc-37630), control siRNA-A (sc-37007), siRNA
(sc-36868) and horseradish peroxidase-conjugated second-ary antibody (sc-2004) Anti-UCP2 serum (ucp21-s) was obtained from Alpha Diagnostic International (San Anto-nio, TX, USA) Anti-cleaved caspase-3 serum (# 9661) and anti-caspase-3 serum (# 9665) were obtained from Cell Sig-naling Technology (Danvers, MA, USA) RINm5F rat ins-ulinoma cells (ATCC # CRL-11605) and RPMI 1640 medium (ATCC # 30-2001) were purchased from the Amer-ican Type Culture Collection (Manassas, VA, USA) INS-1E cells were provided by M Wheeler (University of Toronto) with permission from C Wollheim (University of
(#V13242) were obtained from Invitrogen (Burlington, Can-ada) Electrophoresis and electroblotting materials were
enhanced chemiluminescence kit was obtained from Amer-sham Biosciences (Piscataway, NJ, USA) Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Penzberg, Germany) Microplates and cell culture flasks were obtained from Corning Incorporated (Corning, NY,
(M22424), anti-His-FITC (C6826), Hoechst 33342 (H3570) and 5-(and-6)-chloromethyl-2¢,7¢-dichlorodihydrofluorescein
A
B
C
D
G1
Fig 6 Cellular distribution of endogenous PHB in RINm5F cells RINm5F cells were incubated for 24 h as described in Fig 2 and then processed for immunocytochemis-try as described in the Materials and methods (n = 3 experiments) (A) Hoechst
33342 (nuclei) staining; (B) Mitofluor Red
589 (mitochondria) staining;
(C) anti-PHB ⁄ anti-rabbit-FITC staining; (D) merge Arrows indicate staining of PHB.
Trang 8diacetate (CM-H2DCFDA; P⁄ N46-0309) were obtained
from Molecular Probes (Burlington, Canada) Ethanol was
obtained from the Pharmaceutical Services of the Health
Sciences Centre (Winnipeg, Canada) Anti-actin serum
(A5441), MTT, ATP bioluminescent assay kit (FL-AA) and
all other chemicals were purchased from Sigma-Aldrich
(Mississauga, Canada) For real-time PCR, Optical 96-well
reaction plates (4306737), Power SYBR green PCR master
mix (4367659) and Optical adhesive film (4311971) were
purchased from Applied Biosystems (Foster City, CA,
USA)
Cell culture and treatment
INS-1E and RINm5F cells were grown in RPMI 1640
med-ium containing 10% fetal bovine serum, 1% penicillin and
medium also contained 50 lm 2-mercaptoethanol Briefly,
cells were subsequently incubated for 24 h with or without ethanol or recombinant human PHB (10 nm) This PHB con-centration was chosen as it corresponds to the half-maximal concentration shown to inhibit insulin-stimulated glucose oxidation and pyruvate carboxylase in adipocytes [29] The ethanol dosage was determined using a dose–response curve
in RINm5F [26] and INS-1E cells (see Results)
PHB siRNA transfection
To transfect with siRNA, INS-1E and RINm5F cells were cultured in antibiotic-free RPMI 1640 medium for 24 h
conflu-ency to increase the transfection efficiconflu-ency The transfection was completed by incubating the cells for 7 h in siRNA transfection medium supplied by the manufacturer without serum and antibiotics According to the manufacturer, the PHB siRNA is a pool of three target-specific 19–25 nucleo-tide siRNAs with the following sequences:
360 CAGCTTCCTCGTATCTACATTCAAGAGATG TAGATACGAGGAAGCTGTTTTT;
TCAATATACGGCAGAATGGTTTTT;
AGAAAGGGCAATCTCTGAGTTTTT
The cells were then washed and replaced in fresh normal growth medium After 24 h, the cells were incubated in the medium with or without ethanol for 24 h Floating (apopto-tic) cells were resuspended by gently swirling the culture medium and harvested by mild centrifugation; attached cells were collected after mild trypsinization [54,55] Cell count-ing was performed uscount-ing a Beckman Coulter Z2 particule count and size analyzer The ratio of floating to attached cells was used as an index of apoptosis
PHB overexpression
INS-1E cells were cultured for 24 h before transfection with
a pCMV6-XL5 vector containing a human PHB clone The
using a FuGENE HD transfection reagent (Roche Applied Science, Laval, Canada) according to the protocol supplied with the manufacturer’s instructions The cells were then washed at 24 h and replaced in fresh normal growth
MTT assay
For the MTT assay, culture media were replaced by
MTT and the incubation continued for 3 h The
MTT-PHB Vector PHB
Actin
Caspase 3
Actin
Cleaved caspase 3
*
*
0
Vector
Vector_ETOH Vector_H
2
O2 PHB PHB_ETOH PHB_H
2
O2 20
40
60
80
100
120
A
B
C
Fig 7 Effect of overexpression of PHB on ethanol-induced
apopto-sis in INS-1E cells INS-1E cells were transfected with pCMV6-XL5
vector containing the human PHB clone and subsequently
incu-bated with 80 mM ethanol for 24 h (A) A representative western
blot of three experiments showing the PHB protein expression
level after transfection (B) A representative western blot of
cas-pase-3 and cleaved cascas-pase-3 in cell extracts (n = 3 experiments).
(C) MTT reduction after incubation with 80 mM ethanol (ETOH) or
10 lM H2O2, expressed as the mean ± SEM percentage of vector.
*P < 0.05 versus siRNA control.
Trang 9containing medium was removed after 3 h and replaced
with 200 lL dimethyl sulfoxide to dissolve the formazan
The cells were left for 30 min at room temperature The
reduction of MTT to formazan was quantified by
measur-ing the absorbance at 540 and 630 nm usmeasur-ing a Spectra Max
340 plate reader (Molecular Devices, Sunnyvale, CA,
USA) Each experiment was conducted at least three times,
as shown in the Results, and each treatment was conducted
in triplicate
ROS assay
The production of ROS was determined using the
through the cell membrane and is cleaved by intracellular
esterase into its nonfluorescent form
CM-DCF [5-(and-6)-chloromethyl-2¢,7¢-dichlorofluorescein]
in the presence of ROS [56] Briefly, after cell culture and
treatment, the media were replaced by RPMI 1640
fluorescent compound was measured at the excitation length
488 nm and emission length 505 nm using the SpectraMax
Gemini XS fluorescence microplate reader with the softmax
Flow cytometry
100 lL annexin-binding buffer and serially stained with
5 lL FITC–annexin V and 1 lL propidium iodide for
15 min at room temperature according to the manufac-turer’s instructions After 15 min, the reaction was stopped
by adding 400 lL annexin-binding buffer The stained cells were immediately analyzed by flow cytometry using a high-speed Beckman Coulter EPICS ALTRA flow cytometer (Beckman Coulter Canada Inc., Mississauga, Canada)
cells Histograms were acquired and analyzed using the expo 32 multi comp mfa software, version 1.2B, supplied with the instrument
ATP measurement
The cellular ATP concentration was measured using an ATP bioluminescent assay kit according to the manufac-turer’s instructions The calibration curve was generated
100 lL luciferase assay reagent in disposable polystyrene
siRNA
control
siRNA control_ETOH siRNA PHB
siRNA PHB_ETOH
Caspase 3
Actin
Cleaved caspase 3
siRNA control siRNA control_ETOHsiRNA PHB siRNA PHB_ETOH
Caspase 3
Actin Cleaved caspase 3
PHB
Actin
siRNA Control siRNA PHB
0
50
100
150
200
250
300
** # $
siRNA Control siRNA Control_ETOH siRNA PHB siRNA PHB_ETOH
PHB Actin
siRNA Control siRNA PHB
C
E
D
Fig 8 Effect of PHB siRNA on ethanol-induced apoptosis in RINm5F and INS-1E cells RINm5F (A,C,E) and INS-1E (B,D) cells were transfected with PHB gene siRNA or control siRNA and subsequently incubated with 80 mM ethanol for 24 h Representa-tive western blot of PHB protein expression (A,B) and caspase-3 (C,D) after siRNA trans-fection in RINm5F (A,C) and INS-1E (B,D) cells (E) Percentage ratio of float-ing ⁄ attached cells expressed as the mean ± SEM *P < 0.05 versus siRNA control, **P < 0.01 versus siRNA control,
#P < 0.05 versus siRNA control_EtOH,
$P = 0.056 versus PHB siRNA For each condition, n = 3 experiments.
Trang 10tubes, and incubated at room temperature for 3 min The
light produced was immediately measured for 30 s with an
LB 9507 Lumat luminometer (Berthold, Bad Wildbad,
Germany)
Protein extraction
To prepare the total protein fraction, culture media were
removed and the cells were incubated with 0.05%
in 30 lL lysis buffer (1% Igepal, 0.1% SDS, 0.5%
vortexing, the cells were placed on ice for 30 min, and then
20 min The supernatants were collected and stored at –
described previously [57] After incubation with 0.05%
buffer A (10 mm KCl, 0.1 mm EGTA, 0.1 mm EDTA, 1 mm
dithiothreitol, 10 mm Hepes, pH 7.9 and protease inhibitors)
by gently mixing The cells were placed to swell on ice for
15 min, and then 10 lL 1% Igepal was added After
vigor-ous vortexing for 10 s, the homogenates were centrifuged at
resus-pended in 50 lL ice-cold buffer B (0.4 m NaCl, 1 mm
EGTA, 1 mm EDTA, 1 mm dithiothreitol, 20 mm Hepes,
pH 7.9 and protease inhibitors) by gently mixing The
5 min The supernatants (nuclear fractions) were stored as
above
The mitochondria fraction was prepared as described
previously [58] Briefly, cells were collected with 0.05%
homogenized in 100 lL isotonic buffer (25 mm mannitol,
70 mm sucrose, 1 mm dithiothreitol, 1 mm EGTA, 5 mm
Hepes, pH 7.4 and protease inhibitors) The homogenates
nuclear and cell debris The resulting supernatant was
the mitochondrial fraction The mitochondrial fraction was
each fraction was determined by using the Bio-Rad assay
with BSA as the standard
Western blot analysis
nitrocellulose membrane using a semidry blot apparatus
(Trans-Blot SD Cell, Bio-Rad) After transfer, the membrane
was blocked for 1 h at room temperature with 5% nonfat
mem-brane was then rinsed twice with Tris-buffered saline⁄ 0.1%
Tween 20 and incubated with a primary antibody at room temperature for 1 h After further washing, the membrane was incubated with horseradish peroxidase-conjugated sec-ondary antibody for 1 h and washed twice for 7 min with
were detected using the enhanced chemiluminescence detec-tion kit The same membrane was subsequently stripped with
15 mL strip buffer (100 mm 2-mercaptoethanol, 2% SDS,
rep-robed with anti-actin, anti-heat shock protein 60 or anti-his-tone H1 serum as appropriate Quantitative image analysis was performed using NIH image j software to determine the intensity of the individual proteins
Determination of mRNA expression
PHB and actin gene expression was also determined by real-time PCR, using as primers: PHB: 5¢-GATTTACAG ACAGTGGTGCACACA-3¢ (forward), 5¢-GGGTTCGTAT GGCTGGAAAA-3¢ (reverse); actin: 5¢-AGGGAAATCG TGCGTGACAT-3¢ (forward), 5¢-GAACCGCTCATTGCC GATAG -3¢ (reverse) The cDNA was synthesized with
1 lg total RNA using SuperScriptII RNaseH reverse trans-criptase and random primer (Invitrogen) The primers used
in real-time PCR were designed using primer express soft-ware (version 3.0, Applied Biosystems) The reactions were performed in triplicate under the following conditions:
for 40 cycles Data were analyzed by the DDCt method using abi 7500 system software, and mRNA levels were normalized to actin mRNA
Immunocytochemistry
For immunostaining, RINm5F cells were cultured on chamber slides (Nalge Nunc International, Tokyo, Japan)
in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin until 70–
incubated for 24 h with or without ethanol (80 mm) in the presence of glucose (5.5 or 25 mm) with or without PHB (10 nm) To detect exogenous His-tagged PHB, the cells were fixed with 4% paraformaldehyde for 30 min and
cells were then serially incubated with the mitochondrial
for 20 min, anti-His-FITC (1 : 650) for 1 h and Hoechst
detect the distribution of endogenous PHB, the cells were
Tween 20 for 1 h They were then serially incubated with
589 for 20 min, anti-rabbit FITC (1 : 650) for 1 h and Hoechst 33342 for 5 min The cells were then examined under a Nikon Eclipse TE2000-E fluorescence microscope