Impaired mitochondrial calcium uptake caused by tacrolimus underlies beta-cell failure
Lombardi et al. Cell Communication and Signaling
Impaired mitochondrial calcium uptake caused by tacrolimus underlies beta-cell failure
Angela Lombardi 1
Bruno Trimarco 0
Guido Iaccarino 2
Gaetano Santulli 0 1
0 Department of Advanced Biomedical Sciences, “Federico II” University of Naples , Naples , Italy
1 Department of Medicine, Albert Einstein College of Medicine , New York, NY , USA
2 Department of Medicine, Surgery and Dentistry, “Scuola Medica Salernitana”, University of Salerno , Fisciano , Italy
Background: One of the most common side effects of the immunosuppressive drug tacrolimus (FK506) is the increased risk of new-onset diabetes mellitus. However, the molecular mechanisms underlying this association have not been fully clarified. Methods: We studied the effects of the therapeutic dose of tacrolimus on mitochondrial fitness in beta-cells. Results: We demonstrate that tacrolimus impairs glucose-stimulated insulin secretion (GSIS) in beta-cells through a previously unidentified mechanism. Indeed, tacrolimus causes a decrease in mitochondrial Ca2+ uptake, accompanied by altered mitochondrial respiration and reduced ATP production, eventually leading to impaired GSIS. Conclusion: Our observations individuate a new fundamental mechanism responsible for the augmented incidence of diabetes following tacrolimus treatment. Indeed, this drug alters Ca2+ fluxes in mitochondria, thereby compromising metabolism-secretion coupling in beta-cells.
Mitochondrial calcium; ATP; Diabetes; Insulin release; Immunosuppressive regimen; Ca2+ leak
Tacrolimus (also known as fujimycin and FK506) is a
macrolide lactone isolated from Streptomyces tsukubaensis,
currently used as potent immunosuppressant in organ
transplantation to reduce rejection rates [
]. One of its
most common adverse effects is new-onset diabetes
mellitus following transplantation [
], a serious complication
that also increases the risk of infection and cardiovascular
disease . Indeed, a 5-year follow-up study monitoring
patients treated with tacrolimus after transplant revealed an
incidence of diabetes of 41% [
The exact mechanisms underlying the diabetogenic
effects of tacrolimus have not been fully elucidated.
Various studies have suggested that tacrolimus side effects are
attributable to its peripheral action, engendering a
markedly reduced insulin sensitivity [
]. We hypothesize
that tacrolimus at therapeutic dosage has also a direct
detrimental effect on beta-cells, in particular on
mitochondrial Ca2+ dynamics. To test our hypothesis, we evaluated
the specific effects of tacrolimus on beta-cells.
Cell culture and drugs
INS-1 beta-cells (AddexBio, San Diego, CA) were
cultured in a humidified atmosphere (37 °C) containing 5%
CO2 in RPMI-1640 medium, and insulin levels were
determined as previously described and validated by our
]. In some experiments, cells were treated
with glucose (Bio-Techne, Abingdon, UK), L-leucine and
glutamine (both from MyBioSource, San Diego, CA,
USA), KCl (Merck KGaA, Darmstadt Germany) or
tacrolimus (LC Laboratories, Woburn, MA, dissolved in
Cell viability assay
Cell viability was evaluated using the
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay, as described [
Ca2+ imaging experiments were carried out as previously
]. Briefly, to assess mitochondrial
Ca2+, cells were loaded with Rhod-2 AM (3 μM, Thermo
Fisher Scientific, Waltham, MA) at 37 °C for 30 min,
followed by washout and 1 h rest at room temperature
for de-esterification. Because of its delocalized positive
charge, Rhod-2 AM accumulates preferentially within
the mitochondrial matrix, where it is hydrolyzed and
15, 20, 21
]. Fluorescence was detected using a
pass-band filter of 545–625 nm in response to excitation
at 542 nm. Ca2+ mobilization from the ER following
caffeine (10 mM, Biorbyt, Cambridge, UK) stimulation
was assessed loading the cells with Fura-2 acetoxymethyl
(AM) ester (Thermo Fisher Scientific, 5 μM, 15 min,
37 °C), as described [
]; images were obtained using
a dual excitation fluorescence imaging system: changes
in intracellular Ca2+ were reflected in the ratio of
fluorescence emission acquired above 510 nm in response to
excitation at 340 nm and 380 nm. Intracellular Ca2+ leak
was assessed spectrophotometrically in microsomes
obtained from pancreatic beta-cells, as previously
]. Besides the above mentioned indirect
evaluation of ER Ca2+ in response to caffeine, ER Ca2+
content was assessed using the FRET-based camaleon
D1ER (Addgene, Cambridge, MA) [
Mitochondrial respiration was assessed using the Seahorse
Analyzer (Agilent Technologies, Santa Clara, CA, USA),
adding to each well glucose (16.7 mM), oligomycin (1 μM,
Merck KGaA), carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (FCCP, 0.5 μM, Merck KGaA), rotenone
and antimycin A (both 1 μM, Merck KGaA). After
each assay, cells were collected to quantify DNA
using QuantiFluor dsDNA System (Promega,
Madison, WI, USA).
Cytochrome c oxidase activity assay
The cells were permeabilized by freeze-thaw cycle three
times and mixed with detergent solution (0.1% bovine
serum albumin, 250 mM sucrose, 10 mM KH2PO4
2.5 mM laurylmaltoside, all from Merck KGaA). The
enzymatic activity of cytochrome c oxidase was
spectrophotometrically measured at 550 nm.
The genetically encoded sensors PercevalHR and pHRed
(Addgene) were co-transfected in the cells in order to
measure relative intracellular changes in ATP/ADP and
pH, respectively. PercevalHR was excited at 405 nm
(ADP) and 488 nm (ATP) and emission was collected at
530 nm. pHRed was excited using 405 nm and 546 nm
and emission was collected at 630 nm band pass filter.
Since PercevalHR has been reported to be sensitive to
], a correction for pH was performed.
Experiments were performed in a blinded fashion at
least three times, unless otherwise noted. All results are
presented as mean ± SEM. Statistical analysis was
performed via unpaired t test when comparing two
groups (normal distribution was confirmed by
Anderson-Darling test) and one-way ANOVA, followed
by Tukey-Kramer post hoc correction, when comparing
more than two groups. A P value <0.05 was considered
Glucose-induced insulin secretion (GSIS) is reduced following tacrolimus treatment
To evaluate the effects of tacrolimus on beta-cell
function, we measured GSIS in INS-1 beta-cells. In a
dose-response assay, we found that the treatment with
5 nM tacrolimus, which is the average level that has
been measured in the blood of patients undergoing
organ transplantation [
], was sufficient to determine a
significant reduction of GSIS compared with vehicle
(Fig. 1a). The same dose (5 nM) was then tested in a
time-course assay, which revealed that 24-h incubation
markedly impaired GSIS (Fig. 1b). Importantly, tacrolimus
had a significantly detrimental effect on the metabolic
response of beta-cells to leucine and glutamine (Fig. 1c),
strongly suggesting an alteration in mitochondrial
oxidative metabolism. Supporting this view, insulin
secretion in response to cell depolarization obtained via
KCl, thereby bypassing mitochondria, was comparable
between groups (Fig. 1c).
Therapeutic doses of tacrolimus do not affect beta-cell viability
To test the effect of 5 nM tacrolimus on cell viability, we
performed an MTT assay and we did not observe any
significant effect on beta-cell viability (Fig. 1d).
Tacrolimus alters mitochondrial Ca2+ uptake and
mitochondrial respiration in beta-cells
Since mitochondrial Ca2+ has been shown to be a major
determinant of beta-cell function, especially in terms of
generation of metabolic coupling factors [
evaluated the effect of tacrolimus on mitochondrial Ca2+.
We found that tacrolimus significantly impairs
mitochondrial Ca2+ uptake in beta-cells (Fig. 2a-b). Next, we
assessed mitochondrial oxygen dynamics and we
observed a marked decrease in oxygen consumption rate
following incubation with tacrolimus (Fig. 3a-b),
further supporting our hypothesis of a direct effect of
this immunosuppressant on mitochondrial fitness.
Tacrolimus decreases cytochrome c oxidase activity and causes a decrease in ATP/ADP ratio in beta-cells
To further investigate the role of tacrolimus on
mitochondrial function, we first measured the enzymatic
activity of cytochrome c oxidase, a key component of the
electron transport chain; compared with vehicle-treated
cells, we detected a significant reduction in cytochrome
c oxidase activity following incubation with tacrolimus
(Fig. 4a). Then, we evaluated the dynamics of ATP/ADP
levels within the cell, fundamental regulators of
fuelstimulated insulin release [
]. Interestingly, tacrolimus
significantly reduced ATP/ADP ratio in response to
glucose compared with vehicle-treated cells (Fig. 4b-c).
Tacrolimus at therapeutic doses depletes intracellular Ca2+
Given the anatomical and functional connection between
mitochondria and endoplasmic reticulum (ER) [
we tested the effects of tacrolimus on ER Ca2+ dynamics,
and we observed that 24 h incubation with 5 nM
tacrolimus caused significant ER Ca2+ depletion and intracellular
Ca2+ leak, evaluated both indirectly, assessing Ca2+
dynamics in response to caffeine, and directly, using an
ER-targeted probe, with consistent results (Fig. 5).
The main finding of the present study is that tacrolimus
has a detrimental effect on mitochondrial Ca2+ dynamics
and beta-cell function, determining a reduced
mitochondrial Ca2+ uptake and significantly impairing GSIS. Our
results are consistent with the previous demonstration
by Wollheim and colleagues of the essential role played
by mitochondrial Ca2+ in metabolism-secretion coupling
]. Indeed, one of the key roles of Ca2+ within
mitochondria is to serve as a signal for oxidative metabolism,
activating at least three biochemical reactions in the
Krebs cycle, producing reducing equivalents [
thereby accelerating mitochondrial respiration and ATP
Several studies investigating the potential mechanisms
underlying the pathogenesis of new-onset diabetes
following tacrolimus-based treatments had focused on
8, 10, 33–37
]. Alterations in peripheral
insulin-signaling have been also reported in response to
other immunomodulators, including rapamycin,
cyclosporine A, and steroids [
]. In fact, ultrastructural
analysis has revealed that cyclosporine A causes
mitochondrial dysmorphology in renal allograft biopsies [
Herein, we sought instead to test the effects of tacrolimus
in vitro, using the clonal INS-1 beta-cells, which provide
one of the best systems to directly study the
pharmacologic effects of a compound on beta-cell behavior, avoiding
extracellular confounding factors and potential
compensatory mechanisms present in whole organisms or in islet
], especially for the assessment of
mitochondrial Ca2+ dynamics [
]. Of course, we
acknowledge that the lack of experimental evidence in
islets or animal models can also be seen as a
limitation of this work.
We demonstrate here that 5 nM tacrolimus is sufficient
to determine a marked reduction in GSIS, without
affecting cell viability. We decided to test such dose because
this is the average level of drug that has been actually
measured in islet transplant recipients [
]. The lack of
significant effect of therapeutic doses of tacrolimus on cell
viability is consistent with earlier observations in HIT-T15
hamster beta-cells [
]. Instead, when used at high doses
(e.g. 1 μM) tacrolimus does cause apoptosis, as shown ex
vivo in experiments performed on rat islets [
proapoptotic action of tacrolimus following prolonged
exposures (> 24 h) has been also reported [
We and others have previously shown that
mitochondrial function in pancreatic beta-cells and other
tissues is mechanistically related to intracellular Ca2+
15, 28, 31, 32, 51
]. Here we demonstrate that
tacrolimus treatment directly impairs mitochondrial Ca2+
uptake. This phenomenon could explain the reduced
mitochondrial respiration observed in tacrolimus-treated
beta-cells. Indeed, as mentioned above, Ca2+ activates
dehydrogenases in the tricarboxylic acid cycle within the
We also demonstrate here that an impaired
mitochondrial Ca2+ uptake is mechanistically linked to
mitochondrial dysfunction, as shown by the reduced
enzymatic activity of cytochrome c oxidase following
tacrolimus treatment. Our findings are consistent with
the decreased mitochondrial bioenergetics observed in
rat islets incubated with tacrolimus [
tacrolimus has been shown to potentiate
glucolipotoxicity in different models [
]. Crucially, the dynamic
evaluation of ATP/ADP levels confirms that
tacrolimustreated cells generate less ATP compared with
vehicletreated cells, an observation that is in line with earlier
reports suggesting that mitochondrial Ca2+ is essential for
energy production [
]. The decreased insulin release
detected in tacrolimus-treated beta-cells following
stimulation with secretagogues that trigger insulin secretion via
mitochondrial metabolism (e.g. leucine/glutamine), but
not following KCl-induced membrane depolarization,
corroborates this view, strongly indicating a defect in
mitochondrial bioenergetics. Further studies are necessary to
decipher in detail the exact processes linking tacrolimus
pharmacodynamics to the observed effects on ER and
mitochondrial Ca2+ reported here for the first time.
Potential candidates, currently being investigated in our
laboratory, include the immunophilin FK506 binding protein
(FKBP), which forms a complex with tacrolimus [
and ER stress, which has been recently reported in
tacrolimus-treated lymphocytes [
Our findings individuate in impaired mitochondrial Ca2+
uptake a novel mechanism underlying the increased
incidence of diabetes following tacrolimus treatment.
Ca2+: Calcium; ER: Endoplasmic reticulum; FCCP: Carbonyl cyanide
4-(trifluoromethoxy) phenylhydrazone; Fura-2 AM: Fura-2-acetoxymethyl
ester; GSIS: Glucose-stimulated insulin secretion; Rhod2-AM:
1-[2-Amino-5(3-dimethylamino-6- dimethylammonio- 9-xanthenyl)
phenoxy]-2-(2amino-5-methylphenoxy) ethane-N,N,N′,N′-tetra-acetic acid,
We thank Drs. X. Du, TV. McDonald, RN. Kitsis, and Y. Tomer (Albert Einstein
College of Medicine) for helpful discussions.
G.S. is supported by the NIH (R00DK107895) and ES-DRC P30DK20541.
Availability of data and materials
The datasets supporting the conclusions of this article are included within
AL designed and performed experiments, analyzed data and wrote the
paper; BT and GI analyzed data and contributed to discussion; GS conceived
of the project and experimental plans, analyzed data, and wrote the paper.
All authors read and approved the final manuscript.
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