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Diabetologia (2005) 48: 282–289
DOI 10.1007/s00125-004-1627-9
ARTICLE
M. Anello
.
R. Lupi
.
D. Spampinato
.
S. Piro
.
M. Masini
.
U. Boggi
.
S. Del Prato
.
A. M. Rabuazzo
.
F. Purrello
.
P. Marchetti
Functional and morphological alterations of mitochondria
in pancreatic beta cells from type 2 diabetic patients
Received: 27 April 2004 / Accepted: 4 September 2004 / Published online: 15 January 2005
# Springer-Verlag 2005
Abstract Aims/hypothesis: Little information is available
on the insulin release properties of pancreatic islets iso-
lated from type 2 diabetic subjects. Since mitochondria
represent the site where important metabolites that regulate
insulin secretion are generated, we studied insulin release
as well as mitochondrial function and morphology directly
in pancreatic islets isolated from type 2 diabetic patients.
Methods: Islets were prepared by collagenase digestion
and density gradient purification, and insulin secretion in
response to glucose and arginine was assessed by the batch
incubation method. Adenine nucleotides, mitochondrial
membrane potential, the expression of UCP-2, complex I
and complex V of the respiratory chain, and nitrotyrosine
levels were evaluated and correlated with insulin secretion.
Results: Compared to control islets, diabetic islets showed
reduced insulin secretion in response to glucose, and this
defect was associated with lower ATP levels, a lower
ATP/ADP ratio and impaired hyperpolarization of the
mitochondrial membrane. Increased protein expression of
UCP-2, complex I and complex V of the respiratory chain,
and a higher level of nitrotyrosine were also found in type
2 diabetic islets. Morphology studies showed that control
and diabetic beta cells had a similar number of mitochon-
dria; however, mitochondrial density volume was signif-
icantly higher in type 2 diabetic beta cells. Conclusions/
interpretation: In pancreatic beta cells from type 2 dia-
betic subjects, the impaired secretory response to glucose
is associated with a marked alteration of mitochondrial
function and morphology. In particular, UCP-2 expression
is increased (probably due to a condition of fuel overload),
which leads to lower ATP, decreased ATP/ADP ratio, with
consequent reduction of insulin release.
Keywords Adenine nucleotides
.
Insulin secretion
.
Mitochondria
.
Type 2 diabetes
.
UCP-2
Abbreviations ADP: adenosine diphosphate
.
ATP:
adenosine triphosphate
.
BMI: body mass index
.
BSA:
bovine serum albumin
.
FCCP: carbonylcyanide p-
trifluoromethoxyphenylhydrazone
.
KRB: krebs–Ringer
bicarbonate solution
.
ΔΨ
m
: mitochondrial membrane
potential
.
NEFA: non-esterified fatty acid
.
Rh123:
rhodamine-123
.
SI: stimulation index
.
TCA:
trichloracetic acid
.
TMB: tetramethyl-benzidine
.
UCP-2:
uncoupling protein-2
Introduction
Type 2 diabetes mellitus is a metabolic and vascular
disease that has reached epidemic proportions, and rep-
resents a serious health concern. Its prevalence worldwide
is set to increase from its present level of 150 million to
225 million by the end of the decade [1]. Moreover, its
incidence is increasing at an alarming rate also in children
and adolescents [2]. The long-term complications of this
disease carry a crushing burden of morbidity and mortal-
ity, and most type 2 diabetic patients die prematurely from
a cardiovascular event [3–5].
Type 2 diabetes is characterized by defective pancreatic
beta cell insulin release in response to glucose and by
impaired insulin action on its target tissues. The relative
importance of the secretory defects has been recently
outlined by several clinical observations. Insulin resistance
alone is not sufficient to lead to type 2 diabetes in the
absence of a beta cell defect [6–8]. Patients with impaired
M. Anello
.
D. Spampinato
.
S. Piro
.
A. M. Rabuazzo
.
F. Purrello
Internal Medicine, Department of Internal and Specialistic
Medicine, University of Catania, Ospedale Cannizzaro,
Catania, Italy
R. Lupi
.
M. Masini
.
U. Boggi
.
S. Del Prato
.
P. Marchetti
Department of Endocrinology and Metabolism, Metabolic Unit,
University of Pisa,
Pisa, Italy
F. Purrello (*)
Clinica Medica, Ospedale Cannizzaro,
Via Messina 829,
95126 Catania, Italy
e-mail: fpurrello@virgilio.it
Tel.: +39-095-7262053
Fax: +39-095-7262582
glucose tolerance or in the early stages of type 2 diabetes
always present with defects of beta cell secretion [9].
Clinical diabetes develops only when the compensatory
hypersecretion of insulin by the pancreatic beta cell de-
clines [8]. Moreover, as demonstrated in the UKPDS study
[10], type 2 diabetic patients are characterized by a pro-
gressive decline of insulin secretion that becomes more
severe with the increasing duration of the disease.
Conceivably, a more direct assessment of the functional
characteristics of the diabetic beta cell would represent a
better tool for identification of alterations associated with
impaired insulin secretion. However, little information is
available on the insulin release properties of islets isolated
from type 2 diabetic subjects. Pancreatic islets were stud-
ied from seven type 2 diabetic patients (obtained by intra-
operative biopsy) [11]. The authors reported that despite a
marked reduction of glucose-stimulated insulin secretion
in vivo, a normal insulin release was induced by glucose
from the isolated islets, suggesting that extrapancreatic
factors influence beta cell reaction to glucose in type 2
diabetes. Another study investigated insulin secretion func-
tion in pancreatic islets from two type 2 diabetic organ
donors, and found a marked decrease of insulin secretion
during glucose stimulation, although the secretory response
to a combination of leucine and glutamine was less se-
verely affected [12]. In a recent report, islets from diabetic
donors secreted less insulin and exhibited an elevated
threshold for glucose-induced insulin release [13].
The altered insulin secretory pattern might depend on
genetic and/or acquired abnormalities, including the neg-
ative influence of chronic high glucose [14–17] and/or
high non-esterified fatty acids (NEFA) [18–21] plasma
concentrations (gluco- or lipo-toxicity). In normal beta
cells glucose regulates insulin release through its metab-
olism, and mitochondria represent the site where important
metabolites that regulate insulin secretion are generated
[22–24]. Several studies have focused the attention on the
adenine nucleotides as regulators of insulin secretion. In
particular, the increase of ATP/ADP ratio tightly associates
to glucose-induced insulin granule release [25, 26]. In
addition to mitochondrial glucose oxidation, ATP synthe-
sis and ATP/ADP ratio are regulated by uncoupling
protein-2 (UCP-2) expression [27–30]. UCP-2 is a mem-
ber of a family of proteins located in the mitochondrial
inner membrane, which act as proton channels uncoupling
mitochondrial oxidative phosphorylation. By this mecha-
nism, energy is wasted through heat and cellular ATP
synthesis is decreased [31].
The aim of this work was, therefore, to investigate in-
sulin secretion and mitochondrial function and morphology
in human islets from type 2 diabetic patients. We measured
adenine nucleotides, mitochondrial membrane potential,
the expression of UCP-2, complex I and complex V of the
respiratory chain, nitrotyrosine levels, and correlated them
with insulin secretion. Moreover, we studied mitochondrial
ultrastructure. We found distinct differences between dia-
betic and non-diabetic subjects.
Materials and methods
Human islet preparation Pancreatic islets were prepared
by collagenase digestion and density gradient purification,
as previously reported [32, 33]. All protocols were ap-
proved by the local Ethics Committee. For this study, islets
were obtained from 11 non-diabetic human multiorgan do-
nors (age 58±5.4 years, BMI 24.6±1.4 kg/m
2
, mean±SEM),
and from seven type 2 diabetic patients (age 65±6 years,
BMI 27.4±2.2 kg/m
2
, mean±SEM). Mean duration of
clinical diabetes was 5.6±0.6 years; plasma glucose con-
centration, at the time of admission, was 273.3±38.5 mg/dl.
Four diabetic donors were treated with only diet restriction;
two with sulphonylurea treatment; one with both sulpho-
nylurea and metformin. Three diabetic subjects and three
controls were also screened for GAD antibodies, which
resulted negative. Digestion time was similar in control
(38±3 min) and diabetic (36±4 min) islet isolations. At the
end of the isolation procedure, islets were resuspended in
M199 culture medium (containing 5.5 mmol/l glucose),
supplemented with 10% adult bovine serum, antibiotics
(penicillin, 100 U/ml; streptomycin, 100 μg/ml; gentami-
cin, 50 μg/ml; and amphotericin B, 0.25 μg/ml), and cul-
tured at 37°C in 5% CO
2
.
Insulin secretion Insulin secretion studies were performed
by the batch incubation technique as previously described
[33, 34]. Following a 45-min period of incubation at 37°C
in medium containing 3.3 mmol/l glucose, groups of ap-
proximately 30 islets of comparable size were kept at 37°C
for 45 min in Krebs–Ringer bicarbonate solution (KRB),
0.5% albumin, pH 7.4, containing 3.3 mmol/l glucose. At
the end of this period, medium was completely removed,
assayed to measure “basal” insulin secretion, and replaced
with KRB containing either 16.7 mmol/l glucose, or 3.3
mmol/l glucose plus 20 mmol/l arginine. After additional
45-min incubation, medium was removed, and insulin
levels were measured to assess “stimulated” insulin re-
lease. Insulin secretion was expressed as absolute value, as
percent of islet insulin content, and as stimulation index
(SI), i.e. the ratio of stimulated over basal insulin secretion
[35]. Insulin concentrations were measured by a commer-
cially available immunoradiometric assay (Pantec For-
niture Biomediche, Turin, Italy).
Adenine nucleotide measurement Adenine nucleotides were
measured as previously reported [36]. Following islet in-
cubation with 3.3 or 16.7 mmol/l glucose, the experiments
were stopped by the addition of 0.125 ml of trichloracetic
acid (TCA) (Sigma, St. Louis, MO, USA). The extracts were
frozen at −80°C until the day of the assay, which started with
an appropriate further dilution. ATP and ADP were assayed
in triplicate by a luminometric method [37]. To measure total
ATP+ADP, ADP was first converted into ATP. Samples, with
known concentrations of ADP, without ATP, were run in
parallel to check that the transformation was complete. ATP
was measured by the addition of a reagent containing lu-
ciferase and luciferin (Sigma, St. Louis, MO, USA). The
emitted light was measured in a luminometer (Junior LB
283
9509-Berthold Technologies, Germany). To measure only
ATP, the same previously described procedure was fol-
lowed, except that in the first incubation step pyruvate
kinase was lacking. ADP levels were then calculated by
subtracting ATP from the total ATP+ADP. Blanks and ATP
standards were run through the entire procedure, including
the extraction steps.
Mitochondrial membrane potential (ΔΨ
m
) ΔΨ
m
was mea-
sured using rhodamine-123 (Rh123) (Sigma, St. Louis,
MO, USA) as an indicator of mitochondrial membrane po-
tential changes in an islet cell suspension under glucose
stimulation (16.7 mmol/l). Cell were prepared from ∼3,000
human pancreatic islets according to the method described
before [16]. Briefly, islets were transferred to Ca
2+
-free
KRB at 30°C with 1 mmol/l EGTA, 16.5 μg/ml trypsin,
2 μ g/ml DNAse (Boehringer, Mannheim, Germany), and
were gently resuspended with a Pasteur pipette. Cell dis-
sociation was monitored, by observing the suspension with
a microscope. Single cell suspension was then cultured in
M199 medium overnight at 37°C in a 95% O
2
/5% CO
2
atmosphere. Islet cells were then loaded in KRB buffer
containing 3.3 mmol/l glucose and 10 μg/ml Rh123 for 30
min at 37°C. Cells were resuspended in the same buffer
without Rh123 and transferred to a fluorometer (Hitachi
F-2000) cuvette, and the fluorescence excited at 490 nm
was measured at 530 nm, at 37°C with gentle stirring. Re-
sults are expressed as percentage of basal fluorescence (at
3.3 mmol/l glucose).
Determination of nitrotyrosine Nitrotyrosine concentra-
tions were determined in islet cell lysates by an ELISA
method as reported [38]. Briefly, 96-well plates were
coated with 200 μl of standard curve samples (0.166–15
nmol/l) and 1 μg/μl of lysate (65 μl/well) in 0.1 mol/l
carbonate–bicarbonate buffer (135 μl), pH 9.6, overnight
at 4°C. Afterwards, non-specific binding sites were blocked
with 1% BSA in PBS-T (PBS plus 0.05% Tween 20), for
1 h at 37°C and washed with PBS-T. Plates were then
incubated with purified monoclonal anti-nitrotyrosine mouse
IgG for 1 h at 37°C, washed and incubated with peroxi-
dase-conjugated goat anti-mouse IgG secondary antibody
for 45 min at 37°C. Peroxidase reaction product was gen-
erated using tetramethyl-benzidine (TMB) Microwell Per-
oxidase Substrate (Sigma, St. Louis, MO, USA). Plates
were then incubated 5–10 min at room temperature and the
reaction was stopped with 0.5 mol/l H
2
SO
4
, and optical
density read at 492 nm in a microplate reader.
Western blot analysis Uncoupling protein-2 (UCP-2),
NADH-ubiquinone oxidoreductase (complex I), F
1
-ATP-
synthase (complex V) and SREBP-1c protein levels were
measured by western blot analysis. Briefly, groups of ∼300
human islets were homogenized by sonication in SDS-
PAGE sample buffer and equivalent amounts of proteins
were separated on SDS-polyacrylamide gel (Mini-Protean,
Bio-Rad, Hercules, CA, USA) and electrophoretically trans-
ferred onto nitrocellulose membrane (Amersham Pharmacia
Biotech, England). Blotting efficiency as well as the posi-
tion of protein standards was assessed by Ponceau staining.
After blocking, the membranes were incubated with a rabbit
polyclonal anti-UCP-2 antibody (Alpha Diagnostic Inter-
national, San Antonio, TX, USA) at 1:2,000 dilution in
blocking solution, or with a monoclonal anti-NADH-ubi-
quinone oxidoreductase (Molecular Probes, Eugene, OR,
USA) 1:1,000, or with a goat polyclonal anti-F
1
-ATP-
synthase antibody (Santa Cruz Biotechnology, Inc., USA)
1:1,000, or with a monoclonal anti-SREBP-1c (2A4) anti-
body (Santa Cruz Biotechnology, Inc., USA) 1:1,000 dilu-
tion at 4°C, overnight. The membranes were then blotted
with an anti-rabbit (1:2,000) or an anti-mouse (1:5,000) IgG
peroxidase-linked whole antibody (Pierce, Rockford, IL,
USA), or with a monoclonal anti-goat IgG peroxidase con-
jugate (Sigma, St. Louis, MO, USA) diluted 1:10,000, 1 h
at room temperature. Peroxidase activity was detected
using ECL (Amersham Pharmacia Biotech, England).
Electron microscopy evaluation Electron microscopy stud-
ies were performed as previously described [33, 34, 39].
Pancreatic samples were fixed with 2.5% glutaraldehyde in
0.1 mol/l cacodylate buffer, pH 7.4 for 1 h at 4°C. After
rinsing in cacodylate buffer, the tissue was postfixed in 1%
cacodylate buffered osmium tetroxide for 2 h at room tem-
perature, then dehydrated in a graded series of ethanol,
briefly transferred to propylene oxide and embedded in
Epon-Araldite. Ultrathin sections (60–80 nm thick) were
cut with a diamond knife, placed on formvar-carbon coated
copper grids (200 mesh), and stained with uranyl acetate
and lead citrate.
Statistical analysis Data are presented as the mean±SEM.
Statistical significance was assessed by Student’s t-test, or
one-way ANOVA followed by Newman–Keul’s test when
more than two groups were compared. p Values of less
than 0.05 were considered statistically significant.
Results
Insulin secretion As shown in Table 1, glucose (16.7 mmol/
l)-induced insulin release was significantly lower from the
diabetic as compared to non-diabetic islets. Since islets
from diabetic subjects contained 34% less insulin than
control islets (78±4.7 vs. 118±4.2 μU/islet, p<0.01), we
also expressed our data as percent of islet insulin content
(Table 2). Using this method, glucose-induced insulin
secretion was again lower in diabetic islets. Using both
methods, arginine-stimulated insulin release did not differ
significantly between the two groups (Tables 1 and 2).
These experiments, therefore, showed a selective defect of
type 2 diabetes beta cells to release insulin in response to
glucose stimulation.
Measurements of adenine nucleotides content The ATP-
to-ADP ratio, also in human islets, plays a critical role in
glucose-induced beta cell insulin secretion [25]. There-
fore, we measured adenine nucleotide content in pan-
creatic islets from diabetic and non-diabetic subjects, in
284
the presence of basal (3.3 mmol/l) and stimulating (16.7
mmol/l) glucose concentrations (Fig. 1). Islets from dia-
betic subjects had a higher ATP content in basal condition
(14.22±1.58 vs. 9.82±0.31 pmol/μg of islet DNA, n=30
replicates from type 2 diabetic subjects vs. 45 from
controls, p<0.01) and a higher ATP/ADP ratio (Fig. 1).
In response to glucose stimulation, ATP levels signifi-
cantly increased in control islets (from 9.82±0.31 to
16.11±0.27 pmol/μg of islet DNA, p<0.001, but not in
diabetic islets (from 14.22±1.58 to 13.22±1.31 pmol/μg
of islet DNA), the latter being significantly lower than
control (p=0.01). As a consequence, in response to glu-
cose stimulation, the ATP/ADP ratio was lower in dia-
betic subjects than in control group (15.85±0.98 vs. 24.14±
1.77, p<0.001) (Fig. 1).
Mitochondrial membrane potential measurements Since
the energy to drive ATP formation is provided by a proton
gradient across the inner mitochondrial membrane, we
measured glucose-induced changes in mitochondrial mem-
brane potential (ΔΨ
m
)[40]. The Rh123 fluorescence was
recorded in a cell suspension from islets of diabetic and
non-diabetic subjects, as indicated on a graph section. In
control cells when glucose concentration was increased to
16.7 mmol/l, fluorescence decreased (−9.6±0.1%, mean±
SEM, n=5), indicating the glucose-induced hyperpolari-
zation of ΔΨ
m
(Fig. 2a). Cells from diabetic subjects
showed a decreased hyperpolarization of ΔΨ
m
when glu-
cose was raised to 16.7 mmol/l (−6.5±0.54%, mean±SEM,
n=4, p<0.001) (Fig. 2b). The addition of the uncoupler
carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)
1 μmol/l readily depolarised ΔΨ
m
in both the experimental
conditions (Fig. 2).
Mitochondrial protein expression To analyse the expres-
sion of mitochondrial proteins involved in pancreatic beta
cell energy production, we studied, by Western blot, the
levels of the uncoupling protein-2 (UCP-2), and of com-
plex I (NADH-ubiquinone oxidoreductase) and complex
V(F
1
-ATP synthase) of the respiratory chain. We found
that UCP-2 protein levels were significantly increased in
islets from diabetic subjects (+24±6%, mean±SEM, n=4,
p<0.01) compared to control subjects (Fig. 3). We also
Fig. 1 Adenine nucleotide concentrations (ATP in panel a, ADP in
panel b, ATP-to-ADP ratio in panel c). Batches of five islets from
control (white bar) or type 2 diabetic subjects (black bar) were
incubated in 1 ml of KRB medium containing the indicated glucose
concentrations. At the end of incubation (1 h), islets were processed
for measuring adenine nucleotide content. Results are means±SEM
of 45 replicates from five control and 30 replicates from four type 2
diabetic subjects. ***p<0.001 vs 3.3 mmol/l glucose,
§
p<0.05 vs
control,
§§
p<0.01 vs control,
§§§
p<0.001 vs control
Table 2 Insulin secretion as percentage of islet insulin content/45
min, under glucose (Glu) or arginine (Arg) stimulation in pancreatic
islets from control and type 2 diabetic subjects
Glu (3.3
mmol/l)
Glu (16.7
mmol/l)
Arg (20
mmol/l)
Controls (n=11) 1.98±0.14 4.77±0.38a
b
4.82±0.46a
b
Type 2 diabetes
(n=7)
2.8±0.34b
a
3.29±0.41b
a
5.52±0.71a
b
Data are means ± SEM
a
p<0.05 or less vs. 3.3 mmol/l glucose
b
p<0.05 or less vs. controls
Table 1 Insulin secretion (μU/islet/45 min), under glucose (Glu) or arginine (Arg) stimulation in pancreatic islets from control and type 2
diabetic subjects
Glu (3.3 mmol/l) Glu (16.7 mmol/l) SI-Glu Arg (20 mmol/l) SI-Arg
Controls (n=11) 2.34±0.17 5.63±0.45
a
2.5±0.2 5.71±0.54
a
2.4±0.26
Type 2 diabetes (n=7) 2.18±0.26 2.57±0.32
b
1.26±0.19
b
4.3±0.55
a
2.0±0.14
Data are means ± SEM
a
p<0.05 or less vs. 3.3 mmol/l glucose
b
p<0.05 or less vs. controls
SI-Glu Ratio of glucose-stimulated over basal insulin secretion, SI-Arg ratio of arginine-stimulated over basal insulin secretion
285
found that protein expression of both complex I and V of
the respiratory chain were increased in islets from diabetic
subjects (+14±4.5% and +31±13%, n=4, p<0.01) com-
pared to control (Fig. 3). In order to elucidate if up-
regulation of UCP-2 was due to a higher expression of
SREBP-1c, we also measured the levels of this latter
protein and found no differences between diabetic and
control groups (Fig. 4).
Nitrotyrosine levels Respiratory chain activity leads to the
formation of reactive oxygen species. To investigate if the
increased levels of the respiratory chain enzymes in islets
from diabetic subjects might lead to increased oxidative
stress, we measured nitrotyrosine levels. This compound
derives from the reaction of superoxide and nitric oxide,
and is considered a reliable marker of oxidative stress.
Nitrotyrosine levels were respectively 7.2±0.4 and 9.9±0.4
nmol/l in control islets (n=7) and type 2 diabetes islets
(n=6) (p<0.05).
Fig. 2 Glucose-induced mitochondrial membrane potential changes
(ΔΨ
m
). Glucose (16.7 mmol/l) induced hyperpolarisation in human
pancreatic islet cell suspension, from non-diabetic (a) and diabetic
subjects (b). The depolarizing effect of the protonophore FCCP at
the end of each trace is used as control of the ΔΨ
m
integrity. Results
are percentage of basal fluorescence (at 3.3 mmol/l glucose). The
traces represent mean of five separate experiments for control and
four separate experiments for diabetic subjects. c Summary of ΔΨ
m
changes over the basal level in glucose-induced hyperpolarization of
mitochondrial membrane potential in control (white bar) and in type
2 diabetic subjects (black bar). Results are percentage of decrement
under basal fluorescence (at 3.3 mmol/l glucose). ***p<0.001
Fig. 3 A representative Western blot for UCP-2, F
1
-F
0
ATPsynthase
(complex V) and NADH-ubiquinol oxidoreductase (complex I) in
human pancreatic islets from non-diabetic (lane 1) and type 2 dia-
betic subjects (lane 2). Results are mean±SEM of scanning den-
sitometry relative to islet actin of four separate western blots.
*p<0.05, **p<0.01 vs. non-diabetic subjects
Fig. 4 Representative western blot for SREBP-1c in human pan-
creatic islets from non-diabetic (lane 1) and type 2 diabetic subjects
(lane 2)
286
Electron microscopy studies Endocrine cellular composi-
tion from pancreas preparation was 69±4, 22±4 and 9±2%
in controls and 61±3, 25±7 and 14±5% in diabetic patients
(mean±SEM, n=4), for beta, alpha and delta cells, respec-
tively. Cell viability, measured as trypan blue exclusion,
was higher than 90% in both controls (n=5) and diabetic
patients (n=3). As shown in Fig. 5, mitochondria in type 2
diabetes beta cells appeared round-shaped and hypertro-
phic. Compared to control beta cells (n=112 cells, from
three pancreases), type 2 diabetes beta cells (n=108 cells,
from three pancreases) had a similar number of mito-
chondria (12.0±0.9 vs. 12.4±0.6 per microscopy field).
However, the mitochondrial density volume from type 2
diabetic subjects was significantly (p<0.01) higher (4.7
±0.3 ml %) than control beta cells (3.1±0.4 ml %).
Discussion
In islets isolated from the pancreas of seven multiorgan
donors who were affected by type 2 diabetes, we observed
a clearly reduced insulin release in response to glucose,
whereas the secretion of the hormone during stimulation
with the non-fuel secretagogue arginine was only slightly
affected. In order to investigate the basis of this selective
defect, since mitochondrial metabolism, and the subse-
quent rise of ATP and of ATP/ADP ratio plays a central
role in glucose-induced insulin release, we measured
several key steps of the mitochondrial events that lead to
ATP synthesis and correlated them with insulin secretion.
The energy for ATP production is provided by oxidation
of reducing equivalents via the electron-transport chain.
The enzyme complexes I to V are located at the inner
mitochondrial membrane and the flux of electrons along
the respiratory chain establishes the proton gradient, which
generates the membrane potential. Glucose stimulation
results in the transfer of reducing equivalents to the res-
piratory chain, leading to hyperpolarization of the mito-
chondrial membrane (ΔΨ
m
) and generation of ATP. In
islets from diabetic subjects we found that glucose-in-
duced hyperpolarization of the mitochondrial membrane
was reduced. We also found that ATP levels were lower,
at high glucose, and the ATP/ADP ratio was blunted, in
response to glucose stimulation. These defects could con-
ceivably be due to a reduced electron flux through the res-
piratory chain, or to an over-expression of proteins (such
as UCP-2) that tends to diminish the proton gradient gen-
erated by the respiratory chain. To test the first possibility
we measured the protein expression of complex I and
complex V of the respiratory chain and we found an in-
creased expression that makes this hypothesis unlikely. To
test the second possibility, we measured the protein ex-
pression of UCP-2, and we found, indeed, an increased
expression of this protein. UCP-2 is a member of a family
of proteins located in the mitochondrial inner membrane,
which uncouples mitochondrial oxidative phosphoryla-
tion. By this mechanism, energy is wasted through heat
and cellular ATP synthesis is decreased. UCP-2 protein
expression could be activated by an increased formation of
reactive oxygen species [41]. In agreement with this in-
terpretation, in our model we found increased levels of
nitrotyrosine (a marker of oxidative stress) in diabetic is-
lets. According to these data, therefore, it is possible to
suppose that in beta cells from diabetic patients the in-
creased expression of UCP-2 is responsible of the reduced
hyperpolarization of the mitochondrial membrane, lower
ATP levels, ATP/ADP ratio, and eventually, of the reduced
insulin release in response to glucose.
This sequence of events is coherent with several data
obtained in vitro or in animal models, and recently put in
perspective [42]. Increased UCP-2 levels in beta cells are
associated with decreased insulin secretion [43, 44], and
UCP-2 overexpression in rat pancreatic islets has been
shown to inhibit glucose-stimulated insulin secretion by de-
creasing ATP formation [45]. Moreover, in rodent pan-
creatic islets chronically exposed to high glucose or NEFA
glucose-induced impairment of insulin secretion is asso-
ciated with altered mitochondrial function, including over-
expression of the UCP-2 protein and a consequent decrease
of ATP production [46]. In islets from hyperglycaemic
90% pancreatectomized rats [47] or in human islets ex-
posed to high glucose [48], UCP-2 mRNA or protein ex-
pression was increased, in accordance with a decrease of
glucose-induced insulin release. In a tumoral beta cell line,
chronic exposure to high NEFA both reduced insulin
secretion and increased UCP-2 levels by regulating glu-
cose-induced ATP formation [49, 50]. In other reports,
UCP-2 overexpression by enhancing ATP/ADP ratio re-
stores insulin secretion in islets from ZDF rat [51].
Fig. 5 Mitochondrial structure
in pancreatic islets beta cell
from normal (a) and diabetic
subjects (b). White arrow indi-
cates mitochondria. Hashed
arrow indicates insulin granules.
Mitochondrial density volume
was significantly higher in beta
cells from type 2 diabetic sub-
jects compared to control sub-
jects (4.7±0.3 ml % vs. 3.1±0.4
ml %, p<0.01). Magnification
×16,000
287
Further support to the concept that mitochondria in the
diabetic beta cell are in an altered state comes from the
morphological studies we performed. In fact, by electron
microscopy examination, we found that the density vol-
ume of these organelles was significantly higher in type 2
diabetes beta cells than in control cells. Mitochondria
undergo structural changes that parallel their functional
state in both physiological as well as pathological con-
ditions [52], and their enlargement, which can be induced
by various pathological conditions, can be classified into
two categories: the swelling and the formation of mega-
mitochondria [53]. Both situations are considered to be
adaptive processes at the subcellular level to unfavourable
environments. For example, it has been demonstrated that
when cells are exposed to excess amount of free radicals,
the mitochondria become enlarged decreasing the rate of
oxygen consumption and reducing ROS production [54].
These mechanisms are likely to play a role also in diabetes
and in beta cell function. Indeed, evidence has been re-
cently reported of swelling of mitochondria in sural nerve
biopsies from patients with diabetic neuropathy [55]. In
addition, loss of glucose-stimulated insulin secretion from
isolated rat islets was associated with mitochondrial en-
largement [56]. Finally, we have shown that exposure of
human pancreatic islets to cytotoxic cytokines induces
functional and survival beta cell damage, which is ac-
companied by mitochondrial swelling and enlargement
[57, 58].
We observed a selective secretory defect in response to
glucose, whereas the secretion of the hormone during stim-
ulation with the non-fuel secretagogue arginine was only
slightly affected, demonstrating an intrinsic and selective
functional defect of beta cell function in type 2 diabetes. It is
noteworthy that arginine-induced insulin release is largely
independent on ATP synthesis, since this amino acid di-
rectly affects beta cell membrane potential and ion flux.
The present data, the first to our knowledge, indicate
that in pancreatic beta cells from type 2 diabetic subjects,
the impaired secretory response to glucose is associated
with a marked impairment of mitochondrial function. The
presence of excessive fuel availability increases substrate
influx through the metabolic mitochondria pathways,
leading to the generation of large amounts of high-energy
metabolites and reactive oxygen species. Our novel results
suggest that in the presence of such a situation, pancreatic
beta cell UCP-2 expression is increased in humans, which
leads to a lower ATP level and reduced ATP/ADP ratio in
response to glucose, with consequent impairment of insu-
lin release. A better understanding of these mechanisms,
and the discovering of specific molecular targets would
greatly enhance our clinical efficacy in preserving beta cell
function in type 2 diabetic patients.
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