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Hesperetin promotes longevity and delays aging via activation of Cisd2 in naturally aged mice

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Hesperetin promotes longevity and delays aging via activation of Cisd2 in naturally aged mice

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Background The human CISD2 gene is located within a longevity region mapped on chromosome 4q. In mice, Cisd2 levels decrease during natural aging and genetic studies have shown that a high level of Cisd2 prolongs mouse lifespan and healthspan. Here, we evaluate the feasibility of using a Cisd2 activator as an effective way of delaying aging. Methods Hesperetin was identified as a promising Cisd2 activator by herb compound library screening. Hesperetin has no detectable toxicity based on in vitro and in vivo models. Naturally aged mice fed dietary hesperetin were used to investigate the effect of this Cisd2 activator on lifespan prolongation and the amelioration of age-related structural defects and functional decline. Tissue-specific Cisd2 knockout mice were used to study the Cisd2-dependent anti-aging effects of hesperetin. RNA sequencing was used to explore the biological effects of hesperetin on aging. Results Three discoveries are pinpointed. Firstly, hesperetin, a promising Cisd2 activator, when orally administered late in life, enhances Cisd2 expression and prolongs healthspan in old mice. Secondly, hesperetin functions mainly in a Cisd2-dependent manner to ameliorate age-related metabolic decline, body composition changes, glucose dysregulation, and organ senescence. Finally, a youthful transcriptome pattern is regained after hesperetin treatment during old age. Conclusions Our findings indicate that a Cisd2 activator, hesperetin, represents a promising and broadly effective translational approach to slowing down aging and promoting longevity via the activation of Cisd2.
Hesperetin treatment reduces fat and improves glucose homeostasis in old WT mice. A-D Hesperetin treatment for 6 months (from 22-to 28-month old). A, B Representative Micro-CT analysis and quantification of total body fat (volume) and visceral fat (volume) in 3-month WT, 28-month Veh-treated and 28-month hesperetin-treated WT mice (n = 3-10 mice per group). Yellow circles indicate the area of visceral fat for quantification. C, D Representative Micro-CT analysis and quantification of total body lean (volume) and percent of body lean (%) in 3-month WT, 28-month Veh-treated and 28-month hesperetin-treated WT mice (n = 3-10 mice per groups). E Body weight in 3-month WT, 28-month Veh-treated and 28-month hesperetin-treated WT mice (n = 3-10 mice per groups). F, G Basal levels of blood glucose (fasting 6 h), and blood glucose levels at 120 min measured during glucose tolerance test (GTT) after hesperetin treatment for 6 months (from 20.5-to 26.5-month old) in the Veh-treated and hesperetin-treated mice. H The mRNA levels of key enzymes involved in the pathway of glycogenolysis in the livers of 3-month WT, 26-month Veh-treated and 26-month hesperetin-treated WT mice (n = 3 mice per group) after hesperetin treatment for 5 months (from 21-to 26-month old). The mRNA levels were quantified by RNA-seq analysis. I Schematic pathway of the enzymes and metabolites involved in hepatic insulin signaling and glucose metabolism, including glycolysis, glycogen synthesis, gluconeogenesis, and glycogenolysis, in naturally aged Veh-treated and hesperetin-treated WT mice. The nuclear factor kappa-B kinase subunit β (IKKβ) is able to inhibit insulin signaling via phosphorylation of insulin receptor substrate 1. AKT is central to regulating hepatic insulin action and glucose metabolism. Glycolysis: The key enzymes involved in glycolysis are glucokinase (Gck), phosphofructokinase (Pfkl) and pyruvate kinase (Pklr). Glycogen synthesis: The key enzymes involved in glycogen synthesis are glycogen synthase (Gys2) and glycogen branching enzyme (Gbe1). In addition, glycogen synthase kinase 3β (Gsk3β) is able to phosphorylate and inhibit glycogen synthase activity, whereas the protein phosphatase 1 (PP1) is able to dephosphorylate and promote glycogen synthase activity. Gluconeogenesis: The key enzymes involved in gluconeogenesis are pyruvate carboxylase (Pcx), phosphoenolpyruvate carboxykinase (Pck1/Pepck), fructose 1,6-bisphosphatase (Fbp1) and glucose-6-phosphatase (G6pc). Glycogenolysis: The key enzymes involved in glycogenolysis are glycogen debranching enzyme (Agl) and glycogen phosphorylase (Pygl). In addition, protein kinase A alpha (PKAα) is able to phosphorylate and activate the α subunit of phosphorylase kinase (PhKα); subsequently, the activated PhK phosphorylates Pygl to increase its enzymatic activity. Phosphorylase kinase is one of the three main families of Ca 2+ /Calmodulin-dependent protein kinases. Moreover, the δ subunit of phosphorylase kinase (PhKδ) is the endogenous calmodulin, the activity of which is able to be regulated by intracellular Ca 2+ levels. G6P, glucose-6-phosphate; F6P, fructose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; PEP, phosphoenolpyruvate; OAA, oxaloacetate; G1P, glucose 1-phosphate; Pgm2, phosphoglucomutase 2. Results are presented as mean ± SD. *p < 0.05; **p < 0.005; not significant (n.s.). In (B), (D), (E) and (H), the statistical analyses were performed by one-way ANOVA with Bonferroni multiple comparison test. In (F) and (G), the statistical analyses were performed by Student's t test. All the mice used in this study are males. UT untreated
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Yehetal. Journal of Biomedical Science (2022) 29:53
https://doi.org/10.1186/s12929-022-00838-7
RESEARCH
Hesperetin promotes longevity anddelays
aging via activation ofCisd2 innaturally aged
mice
Chi‑Hsiao Yeh1,2,3, Zhao‑Qing Shen4, Tai‑Wen Wang4, Cheng‑Heng Kao5, Yuan‑Chi Teng4, Teng‑Kuang Yeh6,
Chung‑Kuang Lu4,7 and Ting‑Fen Tsai4,8,9*
Abstract
Background: The human CISD2 gene is located within a longevity region mapped on chromosome 4q. In mice,
Cisd2 levels decrease during natural aging and genetic studies have shown that a high level of Cisd2 prolongs mouse
lifespan and healthspan. Here, we evaluate the feasibility of using a Cisd2 activator as an effective way of delaying
aging.
Methods: Hesperetin was identified as a promising Cisd2 activator by herb compound library screening. Hesperetin
has no detectable toxicity based on in vitro and in vivo models. Naturally aged mice fed dietary hesperetin were used
to investigate the effect of this Cisd2 activator on lifespan prolongation and the amelioration of age‑related structural
defects and functional decline. Tissue‑specific Cisd2 knockout mice were used to study the Cisd2‑dependent anti‑
aging effects of hesperetin. RNA sequencing was used to explore the biological effects of hesperetin on aging.
Results: Three discoveries are pinpointed. Firstly, hesperetin, a promising Cisd2 activator, when orally administered
late in life, enhances Cisd2 expression and prolongs healthspan in old mice. Secondly, hesperetin functions mainly in a
Cisd2‑dependent manner to ameliorate age‑related metabolic decline, body composition changes, glucose dysregu‑
lation, and organ senescence. Finally, a youthful transcriptome pattern is regained after hesperetin treatment during
old age.
Conclusions: Our findings indicate that a Cisd2 activator, hesperetin, represents a promising and broadly effective
translational approach to slowing down aging and promoting longevity via the activation of Cisd2.
Keywords: Longevity, Natural aging, Cisd2, Hesperetin
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Background
Aging is a global burden. e demographic transition to
an aging population is occurring globally via longer life
expectancy [1]. Aging is the accumulation of lifelong
molecular and cellular damage that result in progressive
co-morbidities that affect multiple organ systems
together with the co-occurrence of multiple age-associ-
ated diseases, these increase mortality and morbidity [2].
Currently very few pharmacological treatment options
are available to meet the goal of extending a healthy lifes-
pan while concurrently reducing the likelihood of disabil-
ity before death [3].
Mitochondrial dysfunction is a remarkable hallmark
of aging. Many cellular and molecular hallmarks of aging
had been identified and categorized [4]. Specifically
nine candidate hallmarks have been established, namely
Open Access
*Correspondence: tftsai@ym.edu.tw
4 Department of Life Sciences and Institute of Genome Sciences, National
Yang Ming Chiao Tung University, 155 Li‑Nong St., Sec. 2, Peitou, Taipei 11221,
Taiwan
Full list of author information is available at the end of the article
Page 2 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
DNA damage, telomere loss, epigenetic alterations, loss
of proteostasis, dysregulation of nutrient sensing, mito-
chondrial dysfunction, cellular senescence, stem cell
exhaustion, and alterations to intercellular communi-
cation. It is likely that there are extensive interconnec-
tions between these hallmarks of aging. Accordingly, the
experimental amelioration of one particular hallmark is
likely to impinge on the others, and all together they will
determine the aging phenotype.
Although the molecular pathways contributing to aging
are not fully understood, mitochondrial dysfunction does
play a crucial role in driving age-associated pathophysi-
ology [5]. Mitochondrial dysfunction is able to accelerate
aging in mammals [68]. Previous studies have revealed
that preservation of mitochondrial function is a key
player in maintaining the integrity of many organ systems
[9]. In addition, mitochondrial dysfunction, together
with perturbation to metabolic flexibility [10], result in
a decrease in oxygen consumption and lower ATP pro-
duction; these have been observed in older subjects who
have low physical performance [11] and are a mortality
risk factor [12]. Furthermore, mitochondrial dysfunction
during aging alters mitochondrial energy metabolism,
which, in turn, disturbs the nutrient sensing pathways
that regulate glucose homeostasis and insulin sensitiv-
ity. ese impaired cellular responses compromise whole
body energy metabolism and ultimately increase the risks
of age-related disease [10].
Cisd2 is one of the prolongevity genes that medi-
ate lifespan in mammals. Genetically modified mouse
models have revealed prolongevity genes and led to the
discovery of various related pathways that modulate
mammalian lifespan [13, 14]. According to the databases
collected by the Human Aging Genomic Resources, cur-
rently there are eight genes (Bub1b, Cisd2, Klotho, Pawr,
Pparg, Pten, Sirt1, and Sirt6) that have been experimen-
tally proved to effectively decrease and increase lifespan
by genetic knockout and overexpression in mice [15].
Another report from Pedro de Magalhães etal [14] used
the Gompertz mortality rate model [16] to determine
whether previously reported aging-related mouse genes
statistically affect the demographic rate of aging. Interest-
ingly, out of the 30 genes reported to potentially extend
lifespan in mice, only two genes, namely Cisd2 [17] and
hMTH1 [18], were found to have evidence linking them
to slowing aging.
In humans the CISD2 gene (CDGSH Iron Sulfur
Domain 2; synonyms ERIS, Miner1, NAF-1, WFS2, and
ZCD2) is mapped to chromosome 4q22-24 and is located
within a region where a genetic component for human
longevity has been reported [19]. Although the inter-
val on chromosome 4q22-25 has only modest evidence
associating it with human longevity [20], nevertheless,
in mice, we have demonstrated that Cisd2 deficiency
shortens lifespan via premature aging [6]. Notably, the
level of Cisd2 mRNA and protein decreases during
mouse normal aging [21]. Furthermore, we have shown
that a persistently high level of Cisd2 in mice, achieved
by transgenic expression, extends their median and
maximum lifespan without any apparent deleterious side
effects. Moreover, Cisd2 protects mitochondria from
age-associated damage and functional decline, as well
as attenuating age-associated reduction in whole-body
energy metabolism [17]. ese results suggest that Cisd2
is a fundamentally important regulator of lifespan, and
they provide an experimental basis for exploring the can-
didacy of CISD2 in human longevity.
Cisd2 maintains Ca2+ homeostasis and mitochondrial
function in order to mediate lifespan and healthspan. Our
previous studies have revealed that Cisd2 is essential to
maintaining intracellular Ca2+ homeostasis and mito-
chondrial integrity in multiple organ systems [2224].
e Cisd2 protein is located on the endoplasmic reticu-
lum membrane and outer membrane of mitochondria;
these locations may provide a means of endoplasmic
reticulum-mitochondrial crosstalk through electron
transfer in response to redox stimuli [25]. Direct interac-
tion between endoplasmic reticulum and mitochondria
is crucial for Ca2+ transfer and cellular function [26, 27].
In fact, there is increasing evidence linking altered com-
munication between the endoplasmic reticulum and
mitochondria to apoptosis [28], metabolic diseases [29],
and aging [26]. As a proof-of-principle genetic approach,
we have provided evidence demonstrating that in the
heart, Cisd2 deficiency disrupts Ca2+ homeostasis via
dysregulation of Serca2a activity, which results in Ca2+
dysregulation, mitochondrial Ca2+ overload and dysfunc-
tion, thereby impairing cardiac function. Most strikingly,
in Cisd2 transgenic mice, increased Cisd2 expression
delays cardiac aging and ameliorates age-related cardiac
dysfunction [24, 30]. Consistently, an elevated level of
Cisd2 has been found to delay skeletal muscle aging [17],
to slow down liver aging [31] and attenuate liver car-
cinogenesis [23], as well as reducing Alzheimer’s-related
neuronal loss [32, 33]. Collectively, these mouse genetic
studies confirm that Cisd2 is a key player that controls
lifespan and healthspan.
Whether Cisd2 can be targeted pharmaceutically and is
able to be activated by small compounds remains unclear.
In this study, our aim is to evaluate the feasibility of using
a Cisd2 activator as an effective regimen for delaying
aging. We anticipate that the pharmaceutical activation
of Cisd2, whose expression otherwise decreases during
the natural aging of mice, will have therapeutic benefits
in terms of ameliorating age-related functional decline
and structural damage. Such a compound may help to
Page 3 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
slow down aging or even rejuvenate aged tissues in natu-
rally aged mammals, thereby promoting longevity and
extending a healthy lifespan.
Methods
Generation oftheHEK293‑CISD2 reporter cell line
A CISD2 bacterial artificial chromosome (BAC) reporter
clone was constructed from a 102-kb human BAC clone
(CTD-2303J4, Invitrogen, San Diego, CA, USA, #96012)
that contained the intact gene of human CISD2 in its
native chromosomal setting, together with its flank-
ing upstream and downstream regions. is human
BAC clone carries the entire 23.8-kb genomic sequence
of the CISD2 coding region, a 31.3-kb upstream region
and a 46.9-kb downstream region (Additional file1: Fig.
S1A). To generate the CISD2 BAC reporter clone, IRES-
Luc-pA, namely an internal ribosome entry site (IRES),
luciferase (Luc) and a polyA signal (pA), was inserted
into exon 2 of the CISD2 gene (Additional file1: Fig. S1B)
by in vivo recombineering-based method in Escheri-
chia coli [34]. To establish the HEK293-CISD2 reporter
cell line, the linearized CISD2 BAC reporter construct
and pCI-neo plasmid (Promega, Mannheim, Germany,
#E1841) were co-electroporated (250V with capacitance
500F) into the HEK293 cells and selected with 1g/
mL puromycin (Invitrogen, #A11138-03) for 12–14days
in growth medium (DMEM; Gibco, Carlsbad, CA, USA,
#11965) supplemented with 10% fetal bovine serum, 1%
glutamine/penicillin/streptomycin, 1% non-essential
amino acid, and 1 mM sodium pyruvate. e correct
clones for the HEK293-CISD2 reporter system were con-
firmed by PCR and luciferase reporter assay.
Luciferase reporter assay
e HEK293-CISD2 BAC reporter cells were seeded on
a 96-well plate at 2 × 104 cells/well. After 24h of seeding,
the HEK293-CISD2 BAC reporter cells were treated with
different doses of hesperetin (and various other com-
pounds individually) for 24h. After treatment with each
compound, luciferase activity was assayed using a ONE-
GloTM Luciferase Assay System kit (Promega, #E6120)
following the manufacturer’s instructions. e inten-
sity of luminescence was monitored using an Infinite
200 Microplate Reader (Tecan Group Ltd., Männedorf,
Switzerland).
The noble herb compound library
e herb compound library, which contains 780 sam-
ples, was established to systematically screen for CISD2
activators from the 60 noble herbs (Additional file 1:
Table S1) that were described in a traditional Chinese
medicine book, e Divine Husbandman’s Herbal Foun-
dation Canon (神農草經, Shén Nóng Běn Cǎo Jīng,
written between 200 and 250CE) [35]. Briefly, 50 g of
each herb was crushed and extracted with 200mL of 60%
ethanol shaking at 25°C, this was repeated three times.
e extracted solution (600 mL) was partitioned using
CH2Cl2 (600mL), and then dried on a rotary evaporator.
e CH2Cl2 layer and the 60% ethanol layer were further
separated into five fractions using a silica gel-based high
performance liquid chromatography (HPLC) column
or a C18 HPLC column, respectively. e silica gel col-
umn was eluted using 100mL of 100% CH2Cl2, CH2Cl2/
methanol (95/5), CH2Cl2/methanol (9/1), CH2Cl2/meth-
anol (8/2) and 100% methanol in series to give the C1–
C5 fractions. e C18 column was eluted using 200mL
of ddH2O, 30% methanol (aq), 60% methanol (aq), 90%
methanol (aq), and 100% methanol, in series, to give the
M1–M5 fractions. us, for each herb there are 13 frac-
tions including the crude extract, the CH2Cl2 layer, the
60% ethanol layer, C1–C5 fractions, and M1–M5 frac-
tions. Each fraction was dried using a rotary evaporator
and stored at 20°C.
Identication ofCisd2 activators
Initially, sophoricoside and genistein from Sophora
japonica (Additional file1: TableS1) were identified as
Cisd2 activators. Five fractions (CH2Cl2 layer, C2, C3, C5
and M3) from Sophora japonica gave Z-scores >2; and
pure sophoricoside and genistein were obtained from
the precipitate of the CH2Cl2 layer of Sophora japonica
via a bioassay-guided purification. e precipitate of the
CH2Cl2 layer was dissolved in methanol and purified by
semi-preparative reverse phase HPLC (Cosmosil C18
ARII, 10×250mm) under the following conditions: iso-
cratic running 24% acetonitrile/H2O for 20min, then to
60% acetonitrile/H2O in 5min, and hold for 5min; flow
rate at 4mL/min; monitoring at 200 to 400nm range of a
diode array detector. e structures of sophoricoside and
genistein were confirmed by NMR [36, 37] and MS/MS
data. Subsequently, based on the chemical structures of
sophricoside and genistein, seven structural analogs of
these flavonoids, namely baicalin, formononetin, Kaem-
ferol-3-O-rhamnoside, medicarpin, puerarin, rutin, and
hesperetin were selected and examined for their ability
to enhance CISD2 expression using the HEK293-CISD2
reporter cell assay (Additional file1: Fig. S1C, D). Finally,
hesperetin (a single compound with > 98% purity) was
identified as a promising Cisd2 activator that is able
to enhance Cisd2 expression both invitro and in vivo
(Additional file1: Fig. S1E–G).
Analysis ofhesperetin andits conjugated metabolites
e levels of hesperetin, hesperetin-7-O-beta--glu-
curonide (H7G) and hesperetin-7-O-sulfate (H7S) in
the serum and tissues of the mice were quantified by
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Yehetal. Journal of Biomedical Science (2022) 29:53
LC–MS/MS. e mice were fed the dietary hesperetin
supplemented food ad libitum for 4 months until the
day of sacrifice. To synchronize the food intake, the mice
were fasted for 6h (2 p.m. to 8p.m.) and then fed the
hesperetin supplemented food for 2h (8p.m. to 10p.m.).
Sample preparation for the LC–MS/MS assay con-
sisted of 50µL of mouse serum or tissue homogenates
(liver, cardiac muscle or skeletal muscles) being mixed
with 50µL of 250ng/mL of hesperetin-d3(TORONTO
Research Chemicals INC., Toronto, Canada, #H289502).
Hesperetin-d3 is an internal standard of labelled hespere-
tin in which the three hydrogens were replaced by deu-
terium. e mixture was vortexed and then centrifuged
at 15,000×g for 20min in a Beckman Coulter Microfuge
22R Centrifuge at room temperature. e supernatant
was transferred to a clean tube and finally 15µL of the
supernatant was injected onto the LC–MS/MS system.
LC–MS/MS analysis
e chromatographic system consisted of an Agilent
1200 series LC system and an Agilent ZORBAX Eclipse
XDB-C8 column (5µm, 3.0×150mm) interfaced with
a MDS Sciex API4000 tandem mass spectrometer. e
MS/MS ion transitions monitored were m/z 300/9/163.9,
477.0/301.0, 380.9/301.0 and 303.9/163.8 for hesperetin,
H7G, H7S and hesperetin-d3, respectively. A gradient
HPLC method was employed for separation. e mobile
phase A consisted of 10mM ammonium acetate aqueous
solution containing 0.1% formic acid, while the mobile
phase B consisted of acetonitrile. e gradient profile was
as follows (min/%B): 0.0–0.5/10, 0.5–1.2/60, 1.2–3.4/80
and 3.5–5.0/10. e flow rate was set at 1.5mL/min into
the mass spectrometer with the remainder being split off
to waste. e retention times of hesperetin, H7G, H7S,
and hesperetin-d3 were 2.46, 2.07, 2.16, and 2.44 min,
respectively.
Mice andhesperetin treatment
e CISD2 reporter transgenic (TG) mice were gener-
ated as previously described [38]. Briefly, the linearized
CISD2 BAC reporter construct, which carries luciferase
as the reporter and driven by the human CISD2 pro-
moter, was microinjected into the pronuclei of fertilized
eggs obtained from C57BL/6 mice. e Cisd2 mcKO
mice, which carry a Cisd2 KO background specifically in
the skeletal and cardiac muscles, were generated as pre-
viously described [22]. Briefly, mice carrying the Cisd2
floxed allele (Cisd2 f/f) were bred with transgenic mice
carrying the muscle creatine kinase-Cre (MCK-Cre;
JAX006475). After two generations of breeding, Cisd2
mcKO (Cisd2f/f;MCK-Cre) mice were obtained. All the
mice used in this study are males. All mice have a pure
or congenic C57BL/6 background and were housed in
a specific pathogen-free facility with a 12–12 h light–
dark cycle at constant temperature (20–22°C). For the
dietary hesperetin treatment, old wild-type (WT) mice
(19.5mo to 23.5 mo of age) were provided with a diet
(AIN-93G Growth Purified Diet, TestDiet, St. Louis, MO,
USA; Additional file1: TableS2) containing the vehicle
(Veh) (3.04% propylene glycol [w/w]; Sigma-Aldrich,
Munich, Germany, 16033) with or without hesperetin
(0.07% [w/w]; Sigma-Aldrich H4125; purity (HPLC area
%) > 95%; 100mg/kg/day) for 3 to 6months. After these
treatments, the mice were sacrificed using carbon diox-
ide (CO2) inhalation as the method of euthanasia. All
animal protocols were approved by the Institutional Ani-
mal Care and Use Committee of Chang Gung Memorial
Hospital (No. 2017103002 and 2017030901) and National
Yang Ming Chiao Tung University (No. 1040104r). e
animal protocol was designed to respect the associated
guidelines and the 3R principles (Replacement, Reduc-
tion and Refinement) according to the “Animal Protec-
tion Act” of Taiwan.
In vivo imaging system (IVIS) analysis
For the invivo luciferase assay, the luciferase activity in
CISD2 reporter TG mice was measured before and after
dietary hesperetin treatment (100 mg/kg/day provided
in food) using an InVivo Bioluminescence Imaging Sys-
tem (IVIS) (IVIS 50 System, Xenogen Corp., Alameda,
CA, USA). e CISD2 reporter TG mice were injected
intraperitoneally with the substrate -luciferin (150mg/
kg in PBS) and then anesthetized using 2.5% isoflurane in
IVIS 50 System for image acquisition. e luminescent
intensity at the mouse ventral view was analyzed by living
image software 3.2 (IVIS 50 Imaging System, Xenogen
Corp.). e bioluminescent signal is presented as mean
photons/second/centimeter2/steradian (photon/s/cm2/
sr).
Serum biochemical andcomplete blood count (CBC)
analyses
Whole blood samples were collected from the facial vein
or by cardiac puncture at sacrifice. Serum alanine ami-
notransferase, aspartate aminotransferase, blood urea
nitrogen, creatinine, creatine kinase-MB, total choles-
terol, triglyceride, Ca2+, Mg2+, Na+, K+, Cl levels were
monitored by Fuji Dri-Chem 4000i (Fujifilm, Tokyo,
Japan). Whole blood samples were collected from the
facial vein using an EDTA (final concentration 5 mM)
coated tube. e CBC was analyzed using a hematology
analyzer (model ProCyte Dx, IDEXX, Columbus, OH,
USA).
Page 5 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
Whole body composition analysis
Mouse body lean and fat volumes were measured using a
micro-CT scanner (SkyScan 1076, Bruker, Kontich, Bel-
gium). e quantitative results for lean, fat, and visceral
fat in the whole body of the mice were analyzed using the
three-dimensional structure obtained from the micro-CT
and software SkyScan 1076 (Bruker).
Whole‑body metabolic rate
A TSE Calorimetry Module of the LabMaster System
(TSE Systems GmbH, Homburg, Germany) was used
to monitor the oxygen consumption rate (VO2), carbon
dioxide production rate (VCO2), and energy expenditure
(EE) of mice. Individual mice were acclimated for 72h
and then assayed for another 48h using a 12–12h light-
dark cycle (lights on at 8:00a.m.) with adlibitum access
to food and water. e whole-body metabolic rate of each
mouse was measured by indirect calorimetry and then
corrected according to lean mass, which was calculated
as follows: lean mass=lean volume×1.06g/cm3 muscle
density [39].
Oral glucose tolerance test andinsulin tolerance test
For the oral glucose tolerance test, the mice were orally
administrated with glucose water (1.5mg/g) after a 6 h
fasting (9:00a.m. to 15:00p.m.). Blood samples were col-
lected at the indicated time points [6]. e blood glucose
levels were measured using OneTouch Ultra glucose test
strips and a SureStep Brand Meter (LifeScan, Milpitas,
CA, USA). Serum insulin levels were determined by a
mouse insulin ELISA kit (Mercodia, Uppsala, Sweden,
#10-1249-01). For the insulin tolerance test, the mice
were examined after a 2h fasting (9:00a.m. to 11:00a.m.)
and an intraperitoneal injection of insulin (0.75 U/kg)
(Actrapid human regular insulin, Novo Nordisk, Bags-
værd, Denmark).
Rotarod trials
Rotarod trials were used to examine the motor coordi-
nation, balance and exhaustion resistance of the mice
and were conducted using a Rotarod instrument (RT-01,
Singa Technology Corporation, Taipei, Taiwan). e mice
were placed on a rotarod running at different speeds for
the same duration (5min). Mice were pre-trained three
times (5rpm for 5min) before the tests. In the test phase,
the rotating speed was set at 10, 20 and 30 rpm (the
speed up rate was 1rpm/s). e time of falling was auto-
matically recorded by an infrared sensor at the bottom of
the instrument [40].
Transthoracic echocardiography
Cardiac functions were assessed using a VisualSon-
ics VeVo 2100 Imaging System (VisualSonics, Toronto,
Ontario, Canada). Male mice were anesthetized with 1%
isoflurane in 95% O2. Body temperature was maintained
and monitored at 36°C to 37°C on a heated pad (TC-
1000, CWE Inc., Ardmore, PA, USA). Cardiac function
was assessed using a high-frequency 30–50MHz probe,
as described previously [41]. Data analysis was carried
out using VisualSonics software. e personnel respon-
sible for data acquisition were blinded to the animal
groupings.
Electrocardiography (ECG)
Functional testing of the mice’s hearts using ECG was
performed as described previously [24]. e mice were
maintained on a 12:12 h dark–light cycle with lights
switched on at 6:00am. All procedures took place dur-
ing the light phase. Anesthesia was initially induced by
placing the mice for 3–5min in a chamber filled with
3% volume-to-volume isoflurane (Aesica Pharmaceuti-
cals, Hertfordshire, UK). e mice were then positioned
on a warm pad (ALA Scientific Instruments, New York,
NY, USA) that maintained their temperature during ECG
recording. e mice were able to breath freely through a
nose cone. Anesthesia was maintained by inhalation of
1.5% isoflurane. Continuous 5-min ECGs were obtained
using subcutaneous electrodes attached to the four limbs
and recorded via a PowerLab data acquisition system
(model ML866, ADInstruments, Colorado Springs, CO,
USA) and Animal Bio Amp (model ML136, ADInstru-
ments). e ECG analysis was performed in an unbiased
fashion with 1500 beats being analyzed using LabChart7
Pro version 7.3.1 (ADInstruments). Detection and analy-
sis of QTc interval, QRS intervals, Tpeak-Tend intervals
were set to Mouse ECG parameters. e values obtained
were compared statistically by the Mann–Whitney U
test, and a p < 0.05 was accepted as significant.
Western blotting
Skeletal muscle (femoris and gastrocnemius) and cardiac
muscle tissue samples were homogenized using a MagNA
Lyser (Roche, Basel, Switzerland) in RIPA buffer (50mM
Tris at pH 7.4, 150mM NaCl, 1 mM EDTA, 1% Triton
X-100, 0.5% Sodium deoxycholate, 0.1% SDS with com-
plete protease inhibitor and phosphatase inhibitor cock-
tails [Roche, #04693124001]) and then denatured in 2%
SDS sample buffer (50mM Tris at pH 6.8, 100mM Dithi-
othreitol, 2% SDS and 10% glycerol) for 15min at 100°C.
Total protein lysate was separated by SDS-polyacryla-
mide gel electrophoresis (Bio-Rad, Hercules, CA, USA)
and then electro-transferred to a polyvinylidene fluoride
transfer membrane (PerkinElmer, Waltham, MA, USA,
#NEF1002001PK). e membrane was blocked with 5%
(w/v) non-fat dried milk in TBST buffer (25mM Tris at
pH 7.5, 137mM NaCl, 2.7mM KCl and 0.1% Tween-20
Page 6 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
[v/v]) for 1h at room temperature, and then incubated
with a primary antibody for 14–16h at 4°C. e mem-
brane was then washed three times with TBST buffer
before probing with an appropriate secondary antibody
for 1h at room temperature; washing and then detection
by ECL (ermo Fischer Scientific, Waltham, MA, USA,
#34580). e following antibodies were used: Cisd2,
Gapdh (Millipore, Burlington, MA, USA, #MAB374),
Anti-Rabbit IgG HRP Linked (Sigma-Aldrich, #NA934)
and Anti-Mouse IgG HRP Linked (Sigma-Aldrich,
#NA931).
Histopathology andtransmission electron microscopy
(TEM)
Mouse skeletal muscle (femoris and gastrocnemius) and
cardiac muscle tissue samples were harvested and then
fixed with 10% formalin for 14–16h at 4 °C. e sam-
ples were processed using a tissue processor (STP120,
MICROM, Walldorf, Germany) and embedded in paraf-
fin. H&E, Masson’s trichrome and Sirius Red staining of
tissue sections (3–4 m) were carried out by standard
protocols [22]. e TEM was performed as described
previously [24]. In brief, mouse skeletal muscle (gastroc-
nemius) and cardiac muscle tissues were fixed in a TEM
fix buffer (1.5% glutaraldehyde and 1.5% paraformalde-
hyde in 0.1M cacodylate buffer at pH 7.3), post-fixed in
1% OsO4 and 1.5% potassium hexanoferrate and then
tissues were washed in cacodylate and 0.2 M sodium
maleate buffers (pH 6.0) followed by block-stained with
1% uranyl acetate. Following dehydration, the skeletal
muscle (gastrocnemius) tissue and the cardiac muscle
tissue were embedded in Epon (EMS, Hatfield, PA, USA,
#14120) and sectioned for TEM analysis.
Tissue reactive oxygen species (ROS) andreactive nitrogen
species (RNS) levels
ROS and RNS levels were assayed in the skeletal muscle
and cardiac muscle tissue lysates by an invitro ROS/RNS
Assay Kit for quantification of ROS and RNS levels fol-
lowing the manufacturer’s instructions (Cell Biolabs, San
Diego, CA, USA, #STA-347). e fluorescence intensity
of 2ʹ,7ʹ-dichlorodihydrofluorescein (DCF) was monitored
using an Infinite 200 Microplate Reader (Tecan Group
Ltd.).
Tissue RNA isolation, RNA sequencing, andpathway
analysis
Total RNA was isolated from skeletal muscle (gastroc-
nemius), cardiac muscle, and liver tissue using TRI Rea-
gent (Sigma-Aldrich, #T9424) and phenol/chloroform
extraction. e quality of the total RNA was examined
using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Santa Clara, CA, USA); samples with an RNA Integrity
Number higher than 8 were subjected to RNA sequenc-
ing. e RNA sequencing (RNA-seq) was conducted by
the Genome Research Center at National Yang Ming
Chiao Tung University. e dataset was generated to
a depth of at least 20million reads for each sample by
single-end sequencing. After mapping, the unique gene
reads were analyzed as RPKM (reads per kilobase of
exon model per million reads) to assess gene expres-
sion. A total of 6404 and 6231 genes were retained after
filtering to identify expressed genes in the cardiac mus-
cle and skeletal muscle (gastrocnemius) tissues (minimal
counts in RPKM>4 detected in at least 50% of samples),
respectively. e p-values of the gene expressions were
adjusted using the Benjamini–Hochberg method. Dif-
ferentially expressed genes (DEGs) were identified using
a false discovery rate (FDR) cut-off threshold as indi-
cated in the figure legends. DEGs reversed by hesperetin
were analyzed using the following criteria: (1) 26-month
WT-Veh vs. 3-month WT, FDR<0.1; (2) 26-month WT-
hesperetin vs. 26-month WT-Veh, p<0.05, and revers-
ing of 26-month WT-Veh vs. 3-month WT; (3) 26-month
WT-hesperetin vs. 3-month WT, p > 0.05. e DEGs
from the RPKM was loaded into the EZinfo software
package for principal component analysis (PCA, EZinfo
3.0.3 software, Umetrics, Umeå, Sweden). Gene Ontol-
ogy (GO) functional characterization was performed
using the online tools PANTHER (www. panth erdb. org)
and Mouse Genome Informatics (MGI) GO term finder
(www. infor matics. jax. org). e values of the RPKM were
transformed into z-scores and these scores were used to
generate heatmaps using Multi Experiment Viewer 4.9
software (mev.tm4.org).
Statistical analysis
e data are presented as mean ± SD or mean ± SEM, as
described in the figure legends. Comparisons between
two groups were carried out using an unpaired two-tailed
Student’s t test. Comparisons among groups greater than
two were carried out using either one-way or two-way
ANOVA with Bonferroni multiple comparison test as
indicated in the figure legends. e survival rates of the
mice were compared using a log-rank (Mantel–Cox) test;
power analysis (SPSS Statistics 26.0, IBM Corp, Armonk,
NY, USA) revealed that a sample size of 47 animals
(including the no-treatment, Veh and hesperetin groups)
has a power of 0.9608. When analyzing statistical differ-
ences among groups, p < 0.05 was considered significant
using the software Graphpad Prism 6.0 (GraphPad Soft-
ware, San Diego, CA, USA).
Page 7 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
Results
Hesperetin isapromising Cisd2 activator
withnodetectable toxicity
e beneficial effects of Cisd2 on slowing down aging in
mice have prompted us to translate the genetic evidence
into a pharmaceutical application. To this end, we estab-
lished a HEK293-CISD2 reporter cell line and a trans-
genic (TG) reporter mouse model using the human BAC
(bacterial artificial chromosome) clone CTD-2303J4
that contains the intact gene of human CISD2, in order
to systematically screen for CISD2 activators capable of
enhancing CISD2 expression at the transcriptional level
(Additional file1: Fig. S1A, B). We screened a herb com-
pound library that had been established by our team
from 60 noble herbs (Additional file1: TableS1); these
herbs were described in a traditional Chinese medicine
handbook, e Divine Husbandman’s Herbal Foundation
Canon [35]. After substantial fractionation and charac-
terization, as well as bioassay-guided analyses of various
structural analogs of the identified flavonoids, we identi-
fied hesperetin (a single compound with > 98% purity) as
a promising Cisd2 activator that is able to enhance Cisd2
expression both invitro and invivo (Additional file1: Fig.
S1C–G).
Importantly, hesperetin has no detectable toxicity
under the conditions that are effective in HEK293 cells
(10–30M) and in WT young and old mice. We provided
hesperetin in the mice’s food (100mg/kg/day) in order to
treat naturally aged mice during old age. e dose used in
this study is an achievable dose in humans and is a human
equivalent dose of 491mg/60kg/day based on an equa-
tion developed for interspecies dose conversion from ani-
mal to human studies [42]. After 6months of treatment
of the old WT mice (aged from 20- to 26-month old),
serum biochemical analyses revealed that hesperetin has
no detectable toxicity compared with a sex-matched and
age-matched control group treated with Veh. e invivo
serum parameters analyzed included (1) damage related
to liver, kidney, and cardiac functions; (2) metabolic indi-
ces for serum insulin, total cholesterol, and triglycerides;
and (3) various electrolytes, namely Ca2+, Mg2+, Na+, K+,
and Cl ions. Interestingly, hesperetin seems to decrease
a liver injury marker, namely aspartate aminotransferase
(Additional file1: Fig. S2). Additionally, a CBC analysis
revealed that hesperetin has no detectable toxicity on
hematological parameters after 7months of treatment of
old WT mice (Additional file1: Fig. S3). Taken together,
these serum biochemical and CBC analyses confirm that
hesperetin has no detectable toxicity when used for the
long-term treatment of old mice.
Furthermore, we have quantified the compound con-
centrations of hesperetin and two of its major conju-
gated metabolites, namely H7G and H7S, in the serum,
liver, skeletal muscle and heart of the dietary hesperetin-
treated mice (Additional file1: Fig. S4A–D). In the serum
and tissues examined, all of the three compounds (hes-
peretin, H7G and H7S) are detectable. Interestingly, both
hesperetin (10 M) and a higher concentration of H7S
(30M) are able to activate the CISD2 reporter to a simi-
lar level in the HEK293-CISD2 reporter cells (Additional
file1: Fig. S4E). Accordingly, the anti-aging effects of die-
tary hesperetin could be a combined activity of hespere-
tin and its conjugated metabolites, such as the H7S.
Hesperetin delays aging andpromotes longevity
innaturally aged WT mice
To investigate if hesperetin is able to slow down aging
and extend a healthy lifespan, we treated naturally aged
mice started at 21-month old with dietary hesperetin and
monitored their survival rate. Intriguingly, hesperetin sig-
nificantly extends the lifespan of the aging mice (Fig.1A).
e median lifespan of the hesperetin-treated WT mice
was increased by 2.25months (8.7%; from 25.95- to 28.2-
month old) relative to Veh-treated WT mice (p=0.04),
with the maximum lifespan increase being 4.1 months
(13.9%; from 29.5- to 33.6-month old). e level of Cisd2
was found to be significantly decreased in the skeletal
(femoris and gastrocnemius) and cardiac muscles at
26-month in the untreated old mice. Remarkably, die-
tary hesperetin increases Cisd2 levels in aged tissues to
a level comparable to that of young mice at 3-month old
(Fig.1B–D). ese results show that Cisd2 is able to be
targeted pharmaceutically and that it is activated by die-
tary hesperetin during the late stage of life. As a result of
the enhanced level of Cisd2, there appears to be a delay in
aging and a prolongation of the lifespan among naturally
aged mice.
Hesperetin attenuates whole‑body metabolic decline
inold mice
e hallmarks of aging are tightly associated with meta-
bolic alterations [43]. Decreased whole-body metabo-
lism, increased body fat accumulation, loss of muscle
mass and impaired glucose homeostasis are all observed
during aging [44]. To study the anti-aging and beneficial
effects of hesperetin on age-related metabolic decline,
we examined the whole-body energy metabolism and
body composition of the old mice treated with hespere-
tin. Our results reveal that the VO2, CO2 and EE of Veh-
treated old (28-month old) WT mice were significantly
decreased compared to young (3-month old) WT mice.
Importantly and intriguingly, hesperetin treatment for
6 months attenuates these age-related reductions that
affect the whole-body metabolism of the old mice during
their dark period (Fig.2A–C). When compare over the
Page 8 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
duration of the treatment, among the hesperetin-treated
group, the older 28-month mice (hesperetin treatment
for 6 months) seem to show a trend towards a higher
metabolic rate than that among the 23.5-month mice
(hesperetin treatment for 1.5months) (Additional file1:
Fig. S5). Taken together, these metabolic analyses reveal
that hesperetin appears to attenuate the age-related met-
abolic decline of old mice.
Hesperetin reduces fat andimproves glucose homeostasis
inold mice
Body composition analysis, in particular the fat and mus-
cle present in the whole body, can be used to evaluate
physical fitness. Interestingly, when body composition
was investigated, our results reveal that total body fat and
visceral fat are significantly increased in Veh-treated old
(28-month) WT mice compared with young (3-month)
WT mice. Notably, dietary hesperetin treatment for
6months attenuates this age-related body fat and visceral
fat accumulation (Fig.3A, B). Moreover, hesperetin treat-
ment also attenuated age-related muscle loss; specifically,
the percentage of bodily lean mass is significantly higher
in the hesperetin-treated old mice compared with that in
the Veh-treated control group (Fig.3C, D). However, no
significant difference in body weight was found (Fig.3E).
Furthermore, impaired glucose homeostasis and insu-
lin resistant are also hallmarks of age-related metabolic
alterations and are highly associated with the age-related
decreases in skeletal muscle mass [44]. To study the ben-
eficial effect of hesperetin on glucose homeostasis in old
mice, we perform glucose tolerance tests (GTTs) and
insulin tolerance tests (ITTs). ere is no significant dif-
ference in the GTTs and ITTs between the Veh-treated
and hesperetin-treated old mice (Additional file 1: Fig.
S6); however, there seems to be a trend towards bet-
ter GTT performance among the hesperetin-treated old
mice. In addition, a previous study has revealed that, in
humans, there is a positive correlation between aging
and progressive elevation of blood glucose, including
fasting and 120-min of GTT blood glucose levels [45].
In our mouse study, notably, after hesperetin treatment
for 6months, there was in a significant decrease in both
fasting (6h) and 120-min of GTT blood glucose levels
(Fig.3F, G).
To study the potential mechanism involved, we per-
formed hepatic transcriptome RNA sequencing analy-
sis in order to examine the expression levels of the key
genes involved in the insulin signaling pathway and in the
regulation of glucose homeostasis, namely the pathways
of glycolysis, gluconeogenesis, glycogen synthesis, and
Fig. 1 Hesperetin extends lifespan and enhances Cisd2 levels in old WT mice. A The survival rate of WT mice without any treatment (n = 20 mice)
and old WT mice treated with vehicle (Veh; n = 8) or hesperetin (Hes; n = 19). For the hesperetin treatment, 21‑month old WT mice were treated
with dietary hesperetin (100 mg/kg/day) or Veh control food. Statistical comparison of the survival curves by log‑rank test: Veh versus hesperetin,
p = 0.04; no treatment WT versus hesperetin, p = 0.029; no treatment WT versus Veh, p = 0.5 (n.s.). BD Elevation of Cisd2 protein levels in multiple
tissues of old mice fed with dietary hesperetin at old age. Western blot of Cisd2 in the femoris (B), gastrocnemius (C), and cardiac muscles (D)
of 26‑month mice treated with dietary hesperetin or Veh control food for 5 months (from 21‑month old). The protein level of Cisd2 in 3‑month
WT mice serves as a young mouse control. Data are presented as mean ± SD. *p < 0.05; **p < 0.005 by one‑way ANOVA with Bonferroni multiple
comparison test. All the mice used in this study are males
Page 9 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
Fig. 2 Hesperetin attenuates whole‑body metabolic decline in old WT mice. Hour‑to‑hour average and quantification of A whole‑body oxygen
consumption (VO2), B CO2 production ( VCO2), and C energy expenditure (EE) during the light and dark periods for 3‑month WT, 28‑month vehicle
(Veh) treated and 28‑month hesperetin (Hes) treated WT mice. The old mice (22‑month old) were treated with dietary hesperetin (100 mg/kg/
day) or Veh control food for 6 months and monitored at 28‑month old. The metabolic rate of an individual mouse was monitored for 48 h. The area
under the curve (AUC) from 20:00 to 02:00 during dark period is quantified. For each mouse, two AUC quantitative values of each metabolic index
calculated from the data of two cycles of 24‑h measurement are presented. Data for the VO2, VCO2, and energy expenditure are normalized to lean
mass. Data are presented as mean ± SEM in the hour‑to‑hour metabolic monitoring. Data for quantification of AUC are presented as mean ± SD.
*p < 0.05; **p < 0.005 by one‑way ANOVA with Bonferroni correction for multiple comparisons. UT untreated
Fig. 3 Hesperetin treatment reduces fat and improves glucose homeostasis in old WT mice. AD Hesperetin treatment for 6 months (from 22‑ to
28‑month old). A, B Representative Micro‑CT analysis and quantification of total body fat (volume) and visceral fat (volume) in 3‑month WT,
28‑month Veh‑treated and 28‑month hesperetin‑treated WT mice (n = 3–10 mice per group). Yellow circles indicate the area of visceral fat for
quantification. C, D Representative Micro‑CT analysis and quantification of total body lean (volume) and percent of body lean (%) in 3‑month WT,
28‑month Veh‑treated and 28‑month hesperetin‑treated WT mice (n = 3–10 mice per groups). E Body weight in 3‑month WT, 28‑month Veh‑treated
and 28‑month hesperetin‑treated WT mice (n = 3–10 mice per groups). F, G Basal levels of blood glucose (fasting 6 h), and blood glucose levels at
120 min measured during glucose tolerance test (GTT) after hesperetin treatment for 6 months (from 20.5‑ to 26.5‑month old) in the Veh‑treated
and hesperetin‑treated mice. H The mRNA levels of key enzymes involved in the pathway of glycogenolysis in the livers of 3‑month WT, 26‑month
Veh‑treated and 26‑month hesperetin‑treated WT mice (n = 3 mice per group) after hesperetin treatment for 5 months (from 21‑ to 26‑month
old). The mRNA levels were quantified by RNA‑seq analysis. I Schematic pathway of the enzymes and metabolites involved in hepatic insulin
signaling and glucose metabolism, including glycolysis, glycogen synthesis, gluconeogenesis, and glycogenolysis, in naturally aged Veh‑treated
and hesperetin‑treated WT mice. The nuclear factor kappa‑B kinase subunit β (IKKβ) is able to inhibit insulin signaling via phosphorylation of insulin
receptor substrate 1. AKT is central to regulating hepatic insulin action and glucose metabolism. Glycolysis: The key enzymes involved in glycolysis
are glucokinase (Gck), phosphofructokinase (Pfkl) and pyruvate kinase (Pklr). Glycogen synthesis: The key enzymes involved in glycogen synthesis
are glycogen synthase (Gys2) and glycogen branching enzyme (Gbe1). In addition, glycogen synthase kinase 3β (Gsk3β) is able to phosphorylate
and inhibit glycogen synthase activity, whereas the protein phosphatase 1 (PP1) is able to dephosphorylate and promote glycogen synthase
activity. Gluconeogenesis: The key enzymes involved in gluconeogenesis are pyruvate carboxylase (Pcx), phosphoenolpyruvate carboxykinase
(Pck1/Pepck), fructose 1,6‑bisphosphatase (Fbp1) and glucose‑6‑phosphatase (G6pc). Glycogenolysis: The key enzymes involved in glycogenolysis
are glycogen debranching enzyme (Agl) and glycogen phosphorylase (Pygl). In addition, protein kinase A alpha (PKAα) is able to phosphorylate
and activate the α subunit of phosphorylase kinase (PhKα); subsequently, the activated PhK phosphorylates Pygl to increase its enzymatic activity.
Phosphorylase kinase is one of the three main families of Ca2+/Calmodulin‑dependent protein kinases. Moreover, the δ subunit of phosphorylase
kinase (PhKδ) is the endogenous calmodulin, the activity of which is able to be regulated by intracellular Ca2+ levels. G6P, glucose‑6‑phosphate;
F6P, fructose 6‑phosphate; F1,6BP, fructose 1,6‑bisphosphate; PEP, phosphoenolpyruvate; OAA, oxaloacetate; G1P, glucose 1‑phosphate; Pgm2,
phosphoglucomutase 2. Results are presented as mean ± SD. *p < 0.05; **p < 0.005; not significant (n.s.). In (B), (D), (E) and (H), the statistical analyses
were performed by one‑way ANOVA with Bonferroni multiple comparison test. In (F) and (G), the statistical analyses were performed by Student’s t
test. All the mice used in this study are males. UT untreated
(See figure on next page.)
Page 10 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
Fig. 3 (See legend on previous page.)
Page 11 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
glycogenolysis. Insulin promotes glycogen synthesis and
inhibits both glycogenolysis and gluconeogenesis via the
regulation of multiple pathways downstream of insulin;
this includes activation in the liver of PI3K/Akt signal-
ing, inhibition of glycogen synthase kinase 3β (GSK3β)
and inhibition of forkhead box protein O1 (Foxo1) [46,
47]. Previous studies have revealed that dysregulation
of hepatic insulin signaling results in impaired glucose
homeostasis and the development of insulin-resistance
during aging [48]. Notably, in the Veh-treated old mice,
most of the DEGs related to insulin signaling pathway
are significantly increased in the livers; most of these
DEGs belong to four major functional groups, namely
inhibition of insulin signaling, glucose metabolism, lipid
metabolism, and cell proliferation/differentiation (Addi-
tional file1: Fig. S7A). Interestingly, in the hesperetin-
treated old mice, the expression of the DEGs related to
insulin signaling pathway and glucose metabolism show
a reversal towards an expression level similar to that of
young mice (Additional file1: Fig. S7B–D). Importantly,
the expression of nuclear factor kappa-B kinase subunit
β (IKKβ), which inhibits insulin signaling and is associ-
ated with hepatic insulin resistance [49], is significantly
increased in the livers of the Veh-treated old mice and
this is reverted by hesperetin treatment (Additional file1:
Fig. S7B), which suggests that hesperetin appears to
improve hepatic insulin sensitivity.
Intriguingly, four key enzymes involved in the hepatic
glycogenolysis, namely glycogen debranching enzyme
(Agl), glycogen phosphorylase (Pygl), protein kinase A
alpha (PKAα), and δ subunit of phosphorylase kinase
(PhKδ), appear to be playing important roles in modu-
lating the levels of blood glucose after hesperetin treat-
ment (Fig. 3I). Agl and Pygl are key enzymes and are
involved in hepatic glycogenolysis to produce glucose.
PKAα is a positive regulator that enhances glycogen-
olysis and it is able to phosphorylate and activate the α
subunit of phosphorylase kinase (PhKα). PhKδ, calmo-
dulin, is encoded by five different genes (Calm1, Calm2,
Calm3, Calm4 and Calm5) in mice; however, the expres-
sion of Calm4 and Calm5 was not detectable in the liver
by RNA sequencing. PhKδ regulates PhK enzyme activ-
ity in a Ca2+-dependent manner [50, 51]. Subsequently,
the activated PhK phosphorylates Pygl to switch on its
enzymatic activity thus increasing the production of glu-
cose (Fig.3I). In the Veh-treated old mice, these four key
enzymes of glycogenolysis (Agl, Pygl, PKAα, and PhKδ)
are significantly increased. Remarkably, in the hespere-
tin-treated old mice, the elevation of these enzymes is
down-regulated and reversed to give a level of expres-
sion comparable to that of the young mice (Fig.3H, I);
as a consequent it seems that the elevation in blood glu-
cose found in old untreated mice is attenuated. A similar
observation indicating that age-related impairment of
hepatic glucose production can be prevented by the sup-
pression of glycogenolysis, thereby down-regulating the
blood glucose, has been reported previously [52].
When glycogen synthesis is explored, there is known
to be two key enzymes that are involved in this process,
namely glycogen synthase (Gys2) and glycogen branching
enzyme (Gbe1) (Fig.3I). Insulin signaling enhances Gys2
activity via activation of protein phosphatase 1 (PP1),
which is encoded by Ppp1ca gene, and by inhibition of
glycogen synthase kinase 3β (Gsk3β) [46]. PP1, which
is a positive regulator that enhances glycogen synthesis.
e latter enzyme can dephosphorylate and this activates
Gys2. However, Gsk3β is a negative regulator and is able
to inhibit glycogen synthesis because it can phosphoryl-
ate and inhibit Gys2. In the Veh-treated old mice, these
key enzymes of glycogen synthesis (Gsk3β, PP1, and
Gbe1) are significantly increased. Remarkably, in the hes-
peretin-treated old mice, their expression is significantly
down-regulated and have reverted to an expression level
comparable to that in the young mice (Fig.3I; Additional
file1: Fig. S7E, F). However, there is no significant differ-
ence in the gluconeogenesis pathway between the livers
of Veh-treated and hesperetin-treated old mice. In sum-
mary, our hepatic transcriptomic analyses reveal that
hesperetin appears to ameliorate age-related dysregu-
lation of insulin signaling in the liver and this improve
glucose homeostasis in the treated old mice via the main-
tenance of normal glycogen metabolism including both
glycogenolysis and glycogen synthesis.
Hesperetin slows downskeletal muscle aging inold mice
Sarcopenia, which is characterized by the degenerative
loss of skeletal muscle mass and strength, is accompa-
nied by mitochondrial degeneration and is a hallmark
of skeletal muscle aging; these changes compromise the
functions of the skeletal muscles [44, 53]. To study the
anti-aging effects of hesperetin on skeletal muscles, we
evaluate the function and structure of skeletal muscles in
the old mice treated with hesperetin. Notably, hesperetin
treatment alleviates the age-related functional decline
of skeletal muscles, as revealed by rotarod tests. Inter-
estingly, the functional analysis by rotarod test revealed
that, in the hesperetin-treated group, the 26-month WT
mice (hesperetin treatment for 6months) were found to
have a better performance compared with the control
WT mice at 26-month old (Veh treatment for 6months)
(Fig.4A). In addition, fibrosis analysis using Masson’s tri-
chrome staining revealed overt degeneration and fibrosis
of the skeletal muscles of the old control WT mice (26-
month old); however, hesperetin was able to alleviate
these age-related histopathological deleterious changes
Page 12 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
Fig. 4 Hesperetin slows down skeletal muscle aging in old WT mice. A The Rotarod tests in old WT mice (n = 4–7 mice) were carried out after
dietary hesperetin (100 mg/kg/day) or vehicle control food treatment for 3 and 6 months (started at 20‑month old). B Masson’s trichrome staining
of femoris and gastrocnemius muscles. The 21‑month old WT mice were treated with dietary hesperetin for 5 months and sacrificed at 26‑month
old. C The representative micrographs showing muscle fibers with an intact and a degenerated morphology in the femoris and gastrocnemius
muscles. D, E Quantification of intact and degenerating muscle fibers in femoris and gastrocnemius. Data are presented as mean ± SD. *p < 0.05;
**p < 0.005 by one‑way ANOVA with Bonferroni multiple comparison test in (D) and (E) or Student’s t test in (A). FH TEM analysis of gastrocnemius
muscle. F Young mice at 3‑month old. G Veh‑treated mice at 24‑month old. Mitochondrial degeneration (MD) and fibrosis (*), which may be caused
by myofibril degeneration and Z‑line breakdown (ZLb), are evident and can be easily detected in the Veh‑treated WT mice at 24‑month old. H
Hesperetin‑treated mice at 24‑month old. The age‑related degeneration appears to be reversed as revealed by the presence of intact Z‑lines (ZLs)
and multiple normal‑sized triads. M, mitochondria; SR, sarcoplasmic reticulum; TC, terminal cisternae of the SR. Scale bars, 500 nm
Page 13 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
of the skeletal muscles (Fig.4B). Furthermore, a signifi-
cant decrease in the percentage of degenerating muscle
fibers were found to be present in both the femoris and
gastrocnemius muscles was observed for the hesperetin-
treated old mice compared to the Veh treated control old
mice (Fig.4C–E).
When we used TEM to analyze the ultrastructure of
skeletal muscles we found disturbance of triad archi-
tecture, which involves a T tubule surrounded by ter-
minal cisterna on either side, as well as degeneration of
mitochondria, in the Veh-treated old mice compared
to young mice at 3-month old. Furthermore, myofibril
degeneration, Z-line breakdown and overt fibrosis, were
also detectable in the Veh-treated old mice (Fig.4F, G).
Strikingly, hesperetin treatment for 3months appears to
attenuate and partially reverse these age-related delete-
rious effects at the ultrastructural level, namely there is
preservation of the triad architecture and sarcomere, as
well as a decrease in mitochondrial degeneration. is
means there is a move toward a younger skeletal muscle
ultrastructural pattern among the hesperetin-treated old
mice (Fig.4H).
Hesperetin slows downcardiac aging inold mice
e incidence and prevalence of heart failure increases
in an aged population and is without effective treatment
[54]. In human elderly individuals, cardiac aging is char-
acterized by the following abnormalities: a decreased
ejection fraction, an increased cardiac performance
index, an increase in arrhythmogenesis, and increased
perivascular fibrosis [5557]. Notably, in aged mice, all
these phenotypes were also present in the hearts of the
Veh-treated WT mice at 24-month old as revealed by
echocardiography (measuring mechanical function),
electrocardiography (ECG, measuring electrical func-
tion), and histopathological analyses (Fig. 5). Specifi-
cally, our results revealed that hesperetin improves the
mechanical function of the aged heart, namely the systolic
ejection fraction and diastolic myocardial performance
index (Fig.5A–C). To study the effects of hesperetin on
age-related arrhythmogenesis and electrical dysregula-
tion, we evaluate a continuous 5-min ECG trace obtained
using old mice under hesperetin treatment. Strikingly,
hesperetin appears to alleviate age-related arrhythmo-
genesis (Fig.5D) and rescue abnormalities affecting the
QT interval (Fig.5E) and Tpeak-Tend interval (Fig.5F)
present in the naturally aged mice. Furthermore, cardiac
perivascular fibrosis was also attenuated in the hespere-
tin treatment aged mice as revealed by Sirius Red/Fast
Green staining of their hearts to detect the presence of
collagen (Fig.5G).
In addition, at the ultrastructural level, hesperetin ame-
liorates age-related deterioration of the intercalated disc
(ICD), the mitochondria, and the sarcomeres present in
cardiac muscle. e ICD is a structure that connects and
synchronizes individual cardiomyocytes and allows them
to work together. We use TEM to examine the integrity
of the three types of cell junctions that make up the ICD,
namely gap junctions, desmosomes, and fascia adherens.
In 3-month WT mice (Fig.5H), the three types of cell
junctions were easily identified. On the other hand, in
24-month WT mice without (Fig.5I) or with Veh treat-
ment (Fig. 5J), severe ultrastructural alterations were
detected. ese include breakdown of the fascia adhe-
rens, extension and fragmentation of the gap junctions,
and partial degeneration of the desmosomes with an
increase in the space between the two membranes of the
ICD. Additionally, degenerated and swollen mitochon-
dria, as well as disorganized and degenerated myofibrils,
were easily detected in 24-month WT mice. Interest-
ingly, after 3months of hesperetin treatment (Fig.5K),
the ultrastructural damage to the aged heart had largely
disappeared and seem to have been reversed resulting in
the ICD features and mitochondria now resembling the
situation observed in the hearts of 3-month WT mice.
Altogether, these results demonstrate that hesperetin,
when given at a late-life stage to naturally aged mice, is
able to improve cardiac electromechanical function and
bring about a delay in cardiac aging.
Fig. 5 Hesperetin slows down cardiac aging in old WT mice. A Representative echocardiography images are shown in different groups of mice
(n 5 per group). B, C Ejection fraction and myocardial performance index obtained from echocardiography analysis. D Representative ECG
tracings and continuous 5‑min waterfall plots recorded following anesthesia of the mice. Representative dysrhythmic ECGs, namely missing beat,
ventricular premature complexes (VPCs), atrioventricular block (AV block), irregular PR interval, and widened QT interval, were found in the old WT
mice or Veh‑treated mice. E, F QT interval and Tpeak‑Tend interval measurements obtained from 5‑min sequential beats of whole ECG tracings
from baseline. G Cardiac perivascular fibrosis is examined by Sirius Red/Fast Green staining of the hearts with the aim of detecting collagen. HK
TEM analysis reveals ultrastructure of the mitochondria, myofibril, and ICD in the cardiac muscle of young mice at 3‑month (H), old WT mice at
24‑month (I), Veh‑treated WT mice at 24‑month (J), and hesperetin‑treated WT mice at 24‑month (K). Overt ultrastructural abnormalities are present
in the cardiac muscles of naturally aged mice or Veh‑treated WT mice at 24‑month old. Myelin figure (MF); FA, fascia adherens; GJ, gap junction;
DS, desmosome; MF, myelin figure; MD, mitochondrial degeneration; myofibril degeneration and disorganization (*). For hesperetin treatment in
this study, old WT mice at 21‑month old are treated with dietary hesperetin (100 mg/kg/day) or Veh control food for 3 months and sacrificed at
24‑month old. Quantified data are presented as mean ± SD and analyzed by one‑way ANOVA with Bonferroni multiple comparison test. *p < 0.05;
**p < 0.005
(See figure on next page.)
Page 14 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
Fig. 5 (See legend on previous page.)
Page 15 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
The anti‑aging eect ofhesperetin ismainly dependent
onCisd2
Importantly, we have found that the beneficial effects
of hesperetin on aging and age-related phenotypes are
largely dependent on Cisd2. We used Cisd2 mcKO
(MCK-Cre; Cisd2f/f) mice, which carry tissue-specific
Cisd2 knockouts affecting their skeletal and cardiac
muscles [22], to study if hesperetin needs the presence
of Cisd2 to bring about its anti-aging effects. e Cisd2
mcKO mice display an overt premature aging phenotype
at 3-month old with a shortened lifespan similar to those
observed in the conventional Cisd2KO mice. In addition,
our previous study revealed that the aging phenotype of
Cisd2 mcKO mice at 3-month old is comparable to that
in the WT mice at 26-month old [22]. Accordingly, we
treated Cisd2 mcKO and WT mice with hesperetin for
4 months starting at 3-month old and sacrificed them
at 7-month old (Additional file1: Fig. S8A). No signifi-
cant difference in the concentration of hesperetin in their
serum and various tissues, namely liver, skeletal muscle,
and cardiac muscle, were found between the WT and
Cisd2 mcKO mice (Additional file 1: Fig. S8B). In the
skeletal muscles of the WT mice, rotarod tests and his-
topathological analysis revealed a normal function and
well-organized structure in their muscles on treatment
with Veh or hesperetin; there was no overt effect of hes-
peretin on these control mice since WT mice at 7-month
old have a normal skeletal muscle phenotype (Additional
file1: Fig. S8C, D). In the skeletal muscles of the Cisd2
mcKO mice, in the absence of Cisd2, hesperetin did not
have a beneficial effect and did not improve muscle func-
tions and nor did it reverse the histopathological dam-
age (Additional file1: Fig. S8C, D). In fact, Cisd2 mcKO
mice displayed overt degenerative loss and occasional
rounded and shrunken fibers in their skeletal muscles
both with and without hesperetin treatment (Additional
file1: Fig. S8D). Consistently, in the hearts of the Cisd2
mcKO mice, in the absence of Cisd2, hesperetin lost its
beneficial effect in terms of improvements related to
electrical dysfunction, namely dysrhythmic ECGs (Addi-
tional file1: Fig. S9A, B), as well as improvements related
to histopathological damage, namely myocardial injury
and fibrosis (Additional file1: Fig. S9C).
Moreover, to differentiate the Cisd2-dependent ver-
sus Cisd2-independent effects of hesperetin, we per-
form transcriptomic analysis by RNA sequencing of the
skeletal muscles from the following four groups of mice:
WT-Hes, WT-Veh, Cisd2 mcKO-Hes and Cisd2 mcKO-
Veh (Fig.6A). DEGs analysis revealed that there are 91
DEGs (62 up-regulated and 29 down-regulated genes)
affected by hesperetin in the WT mice. ese DEGs
can be divided into two groups: Cisd2-dependent genes
(72/91; 79%) and Cisd2-independent genes (19/91; 21%)
(Fig. 6B, C). Accordingly, the majority (79%) of DEGs
influenced by hesperetin are Cisd2-dependent and these
genes lose their differential expression patterns in the
absence of Cisd2. Interestingly, there is indeed a smaller
portion (21%) of the DEGs that are Cisd2-independent;
these genes retain a differential expression pattern in
the Cisd2 mcKO mice (Fig. 6C). Pathway analysis of
these DEGs revealed that metabolism is the main path-
way grouping affected by hesperetin for both the Cisd2-
dependent and Cisd2-independent DEGs (Fig. 6D, E).
Since Cisd2 is essential to maintaining intracellular Ca2+
homeostasis and mitochondrial integrity in mammals,
accordingly it is not surprising that majority (72/91; 79%)
of DEGs influenced by hesperetin are Cisd2-dependent
and that a significant portion of them are associated with
metabolism (53/72; 73.6%) and mitochondrial function
(13/72; 18.1%). In summary, these results provide genetic
and transcriptomic evidence confirming that hesperetin
mainly exerts its anti-aging effects via the activation of
Cisd2, and that it functions primarily in a Cisd2-depend-
ent manner.
Hesperetin treatment results inayounger transcriptome
pattern
In order to gain insights into the biological effects of
hesperetin on aging, we perform RNA sequencing of
heart muscle and of skeletal muscle. ree sets of pair-
wise DEG analyses were performed: (Set-1) 26-month
WT-Veh vs. 3-month WT; (Set-2) 26-month WT-Veh
vs. 26-month WT-Hes; (Set-3) 26-month WT-Hes vs.
3-month WT. A cut-off FDR of less than 0.1 was set as
the significance threshold value. From these three sets of
DEGs, we select genes that were differentially expressed
in Set-1, that is, they showed a significant change in
expression due to spontaneous aging. Using Set-2, we
further selected genes that showed a significant change
in expression but were reverted by hesperetin treatment.
Finally, using Set-3, we further selected to identify those
that were not differentially expressed between 3-month
WT mice and hesperetin-treated 26-month WT mice.
GO classification reveals that most of the DEGs
reverted by hesperetin in aged heart and skeletal mus-
cle are involved in similar biological processes and cel-
lular components (Fig.7A). is indicates the presence
of common altered functional pathways in the cardiac
and skeletal muscles of naturally aged mice. e DEGs
were then further grouped into different age-related
pathways by the MGI GO term finder. In both the heart
(Fig.7B, C) and skeletal muscle (Fig.7D, E), the top five
altered pathways are involved in metabolism, proteo-
stasis, mitochondrial function and oxidative stress, cell
death and senescence, as well as the immune response
and inflammation. Furthermore, the subgroups of the
Page 16 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
top two pathways, namely metabolism (Additional file1:
Fig. S10) and proteostasis (Additional file1: Fig. S11), are
also similar. Additionally, hesperetin decreases the levels
of reactive oxygen species (ROS) and reactive nitrogen
species and shifts the expression patterns of several ROS-
related DEGs in aged heart and skeletal muscle toward
the patterns of young mice (Additional file1: Fig. S12).
Collectively, the above transcriptomic analyses reveal
that hesperetin appears to delay aging and bring about a
younger pattern of gene expression in a range of tissues
of old mice, including cardiac muscle and skeletal muscle.
Discussion
We have shown here that, in old mice, a Cisd2 acti-
vator such as hesperetin is able to act as an effective
translational therapy for slowing down aging and pro-
moting longevity via activation of Cisd2. ree find-
ings are pinpointed by this study. Firstly, Cisd2 is able
to be targeted pharmaceutically and activated by small
Fig. 6 Cisd2‑dependent and Cisd2‑independent DEGs in the skeletal muscles of WT and Cisd2 mcKO mice after hesperetin treatment for 4 months.
A The 3‑month old WT and Cisd2 mcKO mice, which carry a Cisd2KO background specifically in the skeletal and cardiac muscles, were treated
with dietary hesperetin (Hes) (100 mg/kg/day provided in food) or Veh control food (3.04% propylene glycol, w/w) for 4 months and sacrificed at
7‑month old. The transcriptome of skeletal. muscle (gastrocnemius) were analyzed for the following four groups of mice: WT‑hesperetin, WT‑Veh,
Cisd2 mcKO‑hesperetin and Cisd2 mcKO‑Veh. B Principal component analysis (PCA, EZinfo 3.0.3 software) of all the genes affected by hesperetin
in the skeletal muscle of WT mice (91 DEGs, WT‑hesperetin vs WT‑Veh, FDR < 0.2). C Heatmap illustrating that the 91 DEGs (62 up‑regulated and 29
down‑regulated genes) can be divided into two groups: Cisd2‑dependent (72/91; 79%) and Cisd2‑independent (19/91; 21%). Cisd2‑dependent
DEGs: mcKO‑hesperetin vs mcKO‑Veh; p > 0.05. Cisd2‑independent DEGs: mcKO‑hesperetin vs mcKO‑Veh; p < 0.05. D Pathway analysis of the
Cisd2‑dependent DEGs. Metabolism, which is the number one pathway, is sub‑divided into four pathways, namely protein metabolism, nitrogen
metabolism, nucleotide and nucleic acid metabolism, and lipid metabolism. E Pathway analysis of the Cisd2‑independent DEGs. Metabolism, which
is again the main pathway affected by hesperetin, is sub‑divided into three pathways, namely nitrogen metabolism, nucleotide and nucleic acid
metabolism, and lipid metabolism. The grouping of the pathways was carried out by MGI GO term finder (pathway p < 0.05)
Page 17 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
compounds, one being hesperetin. Intriguingly, late-
life treatment with hesperetin of old mice is able to
increase Cisd2 expression to a level comparable to that
of young mice. Leading from this, this enhanced level
of Cisd2 expression appears to delay aging or even
bring about the rejuvenation of aged tissues, thereby
promoting longevity and extending a healthy lifespan.
Secondly, hesperetin exerts many beneficial effects on
various age-related structural defects and functional
declines. Examples identified in old mice include hes-
peretin induced attenuation of age-related whole-body
energy metabolic decline, a decrease in body fat accu-
mulation, an amelioration of body lean loss, and an
improvement in systemic glucose homeostasis. In
addition, hesperetin also delays the aging of the car-
diac and skeletal muscle, which is crucial to systemic
metabolism. Most importantly, hesperetin functions
mainly in a Cisd2-dependent manner and the therapeu-
tic benefits of hesperetin disappear when Cisd2 gene is
absent. irdly, oral administration of hesperetin dur-
ing old age results in a younger transcriptome pattern
that is similar to the pattern found in young mice and is
Fig. 7 Hesperetin treatment results in a younger transcriptome pattern. A A pie chart shows their biological processes and subcellular localization
based on GO annotation. The GO was analyzed using the PANTHER functional classification. B Heatmap illustrating that a total of 141 DEGs are
reversed by dietary hesperetin (126 up‑regulated and 15 down‑regulated genes; 26‑month WT‑Veh vs 3‑month WT, FDR < 0.1) and the aged heart
pattern is moved toward the pattern of a young heart. C The DEGs in the aged heart, which are reverted by hesperetin in panel (B), are grouped
into different age‑related pathways and presented as a percentage. D Heatmap illustrating that all 41 DEGs that are reversed by dietary hesperetin
(9 up‑regulated and 32 down‑regulated genes; 26‑month WT‑Veh vs 3‑month WT, FDR < 0.1) and the aged skeletal muscle (gastrocnemius);
this showing a movement toward the pattern present in young skeletal muscle. E The DEGs in the aged skeletal muscles, which are reverted by
hesperetin in panel (D), are grouped into different age‑related pathways and presented by percentage. In this study, mice were treated with dietary
hesperetin (100 mg/kg/day) or Veh control food from 21‑ to 26‑month. The grouping of the pathways was carried out by MGI GO term finder
(pathway p‑value < 0.05)
Page 18 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
distinctly different to that found in naturally aged mice.
is beneficial or “youthful” profile brought about by
hesperetin includes genes involved in metabolism, pro-
teostasis, mitochondrial function and oxidative stress,
cell death and senescence, as well as immune responses
and inflammation. All of these pathways are associated
with age-related biological processes.
Hesperetin activates Cisd2 toextend healthy lifespan
In humans, several large meta-analyses of human popula-
tions had revealed that antioxidants, folic acid and B vita-
mins, as well as multivitamin and mineral supplements are
ineffective in terms of increasing a healthy lifespan. ere
is no clear evidence in human studies to support the ben-
eficial effect of these supplements in relation to all-cause
mortality, cardiovascular disease, cancer, or cognitive
function [58]. In animal studies using mammalian models,
many studies have shown that treatment with antioxidant
supplements, when aimed at extending lifespan, result in
no overt effect or even can have a negative effect [59].
Remarkably, oral administration of hesperetin, when
begun at a late-life stage, is able to extend healthy lifes-
pan. Hesperetin is a flavanone aglycone found in citrus
fruit peels. Previous studies revealed that hesperetin is
converted into various conjugated metabolites after oral
administration in rats and humans [60, 61]. Moreover,
hesperetin is able to scavenge ROS and reduce lipid per-
oxidation without a cytotoxic effect [62]. Hesperetin is not
only a strong peroxynitrite (ONOO) scavenger, but also
an effective inhibition of peroxynitrite-mediated nitration
of tyrosine through electron donation [63]. In addition,
hesperetin has cancer chemopreventive effects through
a variety of targets related to oxidative and inflamma-
tory processes [64]. Previously studies have also revealed
that a variety of chemically induced cancers, such as uri-
nary bladder, mammary, and colon cancers, are able to
be inhibited by hesperetin based on cellular and animal
models [65]. When metabolic disorders in animal mod-
els are considered, the following effects have been iden-
tified. In the heart, hesperetin in mice is able to inhibit
pressure-overload cardiac remodeling [66], and attenuate
post-infarction cardiac fibrosis through inhibition of the
NF-kB pathway [67]. In the liver of hamsters, food sup-
plemented with hesperetin and its chemical’s metabolites,
3,4-dihydroxyphenylpropionic acid and 3-methoxy-4-hy-
droxycinnamic acid, would seem to be able to inhibit
hepatic 3-hydroxy-3-methylglutaryl-coenzyme reductase
and lowered plasma total cholesterol [68].
Cisd2 activator hesperetin provides analternative toCR
mimetics
Calorie restriction (CR) with adequate nutrition is
the only known non-genetic non-pharmacological
intervention that results in a 20% to 40% increase in small
mammals’ lifespan; it is also the most consistent method
[69]. is approach delays or reduces the risk of many
age-related diseases. e molecular signaling pathways
mediating the anti-aging effect of CR include insulin/
insulin growth factor-1, sirtuins, AMP-activated protein
kinase, and the target of rapamycin, all of which form a
complex interacting network. Several CR mimetics have
been identified, including resveratrol (a SIRT1 activator),
metformin (an AMP kinase activator) and rapamycin (an
mTOR inhibitor). However, trials of these CR mimetics
in healthy humans are currently limited [70]. Chronic
rapamycin use can lead to hepatic gluconeogenesis, insu-
lin resistance, severe glucose intolerance and even diabe-
tes in small animals. Furthermore, 25% to 50% of patients
treated with metformin suffer from poorly tolerated
gastrointestinal side effects. Accordingly, an alternative
regimen to the use of CR mimetics is needed as an inter-
vention in geriatric medicine.
Here, we have successfully translated a genetic concept
into a pharmaceutical approach in which a young-pattern
of Cisd2 expression can be regained by oral administra-
tion of a Cisd2 activator, in this case, hesperetin, which is
safe when used as a long-term supplement in food, even
at the late-life stage of an aging mouse. Hesperetin is able
to retard the aging process and ameliorate age-related
functional declines based on this animal model.
Conclusions
Hesperetin is the first compound we have tested as a
proof-of-concept for the hypothesis that a Cisd2 activa-
tor will have an anti-aging effect. Our findings provide an
experimental basis for using Cisd2 as a molecular target
for the screening and development of novel compounds
that are able to activate Cisd2 pharmaceutically with the
goal of translating these drugs into clinical interventions
that can be used in geriatric medicine. Most importantly,
hesperetin can be rapidly delivered systematically to mul-
tiple organs and tissues invivo. Additionally, it has no
detectable invivo toxicity after long-term oral adminis-
tration for 6–7months in mice, specifically when sup-
plemented in food at a dose of 100mg/kg/day, which has
a human equivalent dose of 491mg/60kg/day. Accord-
ingly, it will be of great interest to develop hesperetin as
a medicinally or nutritionally functional food [68] for
preventive purposes related to extending healthy lifes-
pan and/or therapeutic purpose related to treating age-
related diseases.
Abbreviations
Agl: Glycogen debranching enzyme; BAC: Bacterial artificial chromosome;
CBC: Complete blood count; CR: Calorie restriction; DEG: Differentially
expressed genes; ECG: Electrocardiography; EE: Energy expenditure; FDR: False
Page 19 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
discovery rate; G1P: Glucose 1‑phosphate; G6P: Glucose 6‑phosphate; G6pc:
Glucose‑6‑phosphatase alpha; GO: Gene Ontology; GTT : Glucose tolerance
tests; H7G: Hesperetin‑7‑O‑beta‑d‑glucuronide; H7S: Hesperetin‑7‑O‑sulfate;
Hes: Hesperetin; HPLC: High performance liquid chromatography; ICD: Inter‑
calated disc; ITT: Insulin tolerance tests; mcKO: Skeletal and cardiac muscles
tissue‑specific knockout; MGI: Mouse Genome Informatics; Pgm2: Phospho‑
glucomutase 2; PhK: Phosphorylase kinase; PKAα: Protein kinase A alpha; Pygl:
Glycogen phosphorylase; ROS: Reactive oxygen species; RPKM: Reads per
kilobase of exon model per million reads; TEM: Transmission electron micros‑
copy; TG: Transgenic; VCO2: Carbon dioxide production rate; Veh: Vehicle; VO2:
Oxygen consumption rate; W T: Wild‑type.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12929‑ 022‑ 00838‑7.
Additional le1: TableS1. The 60 noble herbs used to establish the
compound library. TableS2. Diet composition of mouse food. Fig. S1.
CISD2 reporter assay for hesperetin and its structural analogs. Fig. S2.
Serum biochemical analyses revealed that hesperetin has no detect‑
able in vivo toxicity after 6 months of treatment in the old WT mice.
Fig. S3. Complete blood count (CBC) analyses revealed that hesperetin
has no detectable toxicity on hematological parameters after 7 months
of treatment in the old WT mice. Fig. S4. Compound concentrations
of hesperetin, and two of the major conjugated metabolites, namely
hesperetin‑7‑O‑beta‑d‑glucuronide (H7G) and hesperetin‑7‑O‑sulfate
(H7S), in the dietary hesperetin‑treated mice, as well as their bioactivity
to enhance CISD2 expression in the HEK293‑CISD2 reporter cells. Fig. S5.
Comparison of whole body metabolic rate after hesperetin treatment
for 1.5 and 6 months in old WT mice. Fig. S6. No significant difference in
the glucose tolerance test (GTT) and insulin tolerance test (ITT) between
the Veh‑treated and Hes‑treated old mice. Fig. S7. Hesperetin shifts the
expression patterns of several insulin signaling‑related differential expres‑
sion genes (DEGs) in the aged livers toward the patterns of young mice.
Fig. S8. Hesperetin delays skeletal muscle aging in a Cisd2‑dependent
manner. Fig. S9. Hesperetin delays cardiac aging in a Cisd2‑dependent
manner. Fig. S10. Subgroups of metabolism‑related DEGs in the hearts
and skeletal muscles. Fig. S11. Subgroups of proteostasis‑related DEGs in
the hearts and skeletal muscles. Fig. S12. Hesperetin decreases the levels
of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and
shifts the expression patterns of several ROS‑related DEGs in the aged
hearts and skeletal muscles toward the patterns of young mice.
Acknowledgements
We thank Prof. Shu‑Ling Fu and Prof. Chi‑Chang Juan for their inspired discus‑
sions. We also thank the following researchers for their technical assistance:
Dr. Chian‑Feng Chen, Yan‑An Lin, Shao‑Yu Hsiung, Hao‑Chih Hung and Yi‑Ling
Chung. We thank the following core facilities: (1) Microscopy Center at Chang
Gung University and Microscopy Core Laboratory, Chang Gung Memorial
Hospital, Linkou for their TEM assistance; (2) Center for Advanced Molecular
Imaging and Translation, Chang Gung Memorial Hospital, Linkou for the
echocardiography; (3) National RNAi Core Facility at Academia Sinica in Taiwan
for shRNA services; (4) Taiwan Mouse Clinic, Academia Sinica and Taiwan
Animal Consortium; (5) Genomics Center for Clinical and Biotechnological
Applications of National Core Facility for Biopharmaceuticals, Taiwan (MOST
109‑2740‑B‑010‑002) for RNA sequencing.
Author contributions
CHY co‑designed the experiments, analyzed the results and drafted the
manuscript. ZQS, TWW, CHK, YCT, TKY and CKL performed the experiments
and analyzed data. TFT designed the experiments, analyzed and interpreted
the results, and wrote the final manuscript. All authors read and approved the
final manuscript.
Funding
The work was supported by Grants from the Ministry of Science and Technol‑
ogy (MOST107‑2314‑B‑182A‑160‑MY3 and MOST108‑2320‑B‑182A‑003 to
CHY; MOST 107‑3011‑B‑010‑001, MOST 108‑2321‑B‑010‑016 and MOST
109‑2634‑F‑010‑003 to TFT), from Chang Gung Memorial Hospital (CMRPG
2E0081, 2E0082 and 2E0083 to CHY), and from the Ministry of Health and
Welfare (PD‑109‑GP‑02 to TFT).
Availability of data and materials
The data that support the findings of this study are available from the cor‑
responding author upon reasonable request.
Declarations
Ethics approval and consent to participate
All animal protocols were approved by the Institutional Animal Care and
Use Committee of Chang Gung Memorial Hospital (No. 2017103002 and
2017030901) and National Yang Ming Chiao Tung University (No. 1040104r).
Consent for publication
Not applicable.
Competing interests
The authors declare no conflict of interest. TFT and CKL are inventors on the
Taiwan patent TW109120312.
Author details
1 Department of Thoracic and Cardiovascular Surgery, Chang Gung Memorial
Hospital, Linkou, Taoyuan, Taiwan. 2 College of Medicine, Chang Gung Univer‑
sity, Taoyuan, Taiwan. 3 Community Medicine Research Center, Chang Gung
Memorial Hospital, Keelung, Taiwan. 4 Department of Life Sciences and Insti‑
tute of Genome Sciences, National Yang Ming Chiao Tung University, 155
Li‑Nong St., Sec. 2, Peitou, Taipei 11221, Taiwan. 5 Center of General Education,
Chang Gung University, Taoyuan, Taiwan. 6 Institute of Biotechnology and Phar‑
maceutical Research, National Health Research Institutes, Miaoli, Taiwan.
7 National Research Institute of Chinese Medicine, Taipei, Taiwan. 8 Institute
of Molecular and Genomic Medicine, National Health Research Institutes,
Miaoli, Taiwan. 9 Center for Healthy Longevity and Aging Sciences, National
Yang Ming Chiao Tung University, Taipei, Taiwan.
Received: 8 March 2022 Accepted: 19 July 2022
References
1. Beard JR, Officer A, de Carvalho IA, Sadana R, Pot AM, Michel J‑P, et al. The
World report on ageing and health: a policy framework for healthy age‑
ing. Lancet. 2016;387:2145–54.
2. Biesemann N, Ried JS, Ding‑Pfennigdorff D, Dietrich A, Rudolph C, Hahn
S, et al. High throughput screening of mitochondrial bioenergetics in
human differentiated myotubes identifies novel enhancers of muscle
performance in aged mice. Sci Rep. 2018;8:9408.
3. Hansen M, Kennedy BK. Does longer lifespan mean longer healthspan?
Trends Cell Biol. 2016;26:565–8.
4. López‑Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hall‑
marks of aging. Cell. 2013;153:1194–217.
5. Bratic A, Larsson N‑G. The role of mitochondria in aging. J Clin Invest.
2013;123:951–7.
6. Chen YF, Kao CH, Chen YT, Wang CH, Wu CY, Tsai CY, et al. Cisd2 deficiency
drives premature aging and causes mitochondria‑mediated defects in
mice. Genes Dev. 2009;23:1183–94.
7. Sun N, Youle RJ, Finkel T. The mitochondrial basis of aging. Mol Cell.
2016;61:654–66.
8. Wang CH, Kao CH, Chen YF, Wei YH, Tsai TF. Cisd2 mediates lifespan: is
there an interconnection among Ca(2)(+) homeostasis, autophagy, and
lifespan? Free Radic Res. 2014;48:1109–14.
9. Murphy E, Ardehali H, Balaban Robert S, DiLisa F, Dorn Gerald W, Kitsis
Richard N, et al. Mitochondrial function, biology, and role in disease. Circ
Res. 2016;118:1960–91.
10. Riera CE, Dillin A. Tipping the metabolic scales towards increased longev‑
ity in mammals. Nat Cell Biol. 2015;17:196.
11. Distefano G, Standley RA, Zhang X, Carnero EA, Yi F, Cornnell HH,
et al. Physical activity unveils the relationship between mitochondrial
Page 20 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
energetics, muscle quality, and physical function in older adults. J
Cachexia Sarcopenia Muscle. 2018;9:279–94.
12. Stenholm S, Koster A, Valkeinen H, Patel KV, Bandinelli S, Guralnik JM, et al.
Association of physical activity history with physical function and mortal‑
ity in old age. J Gerontol A Biol Sci Med Sci. 2016;71:496–501.
13. Barardo D, Thornton D, Thoppil H, Walsh M, Sharifi S, Ferreira S, et al. The
DrugAge database of aging‑related drugs. Aging Cell. 2017;16:594–7.
14. Pedro de Magalhaes J, Thompson L, de Lima I, Gaskill D, Li X, Thornton D,
et al. A reassessment of genes modulating aging in mice using demo‑
graphic measurements of the rate of aging. Genetics. 2018;208:1617–30.
15. Human Aging Genomic Resources. http:// genom ics. senes cence. info/
genes/ search. php? search= & show= 5& sort= 1& organ ism= Mus+ muscu
lus& long_ influ ence= pro& lifes pan_ effect= incre ase_ decre ase& search= &
page=1. Accessed on 16 May 2022.
16. Finch CE, Pike MC. Maximum life span predictions from the Gompertz
mortality model. J Gerontol A Biol Sci Med Sci. 1996;51:B183–94.
17. Wu CY, Chen YF, Wang CH, Kao CH, Zhuang HW, Chen CC, et al. A per‑
sistent level of Cisd2 extends healthy lifespan and delays aging in mice.
Hum Mol Genet. 2012;21:3956–68.
18. De Luca G, Ventura I, Sanghez V, Russo MT, Ajmone‑Cat MA, Cacci E,
et al. Prolonged lifespan with enhanced exploratory behavior in mice
overexpressing the oxidized nucleoside triphosphatase hMTH1. Aging
Cell. 2013;12:695–705.
19. Puca AA, Daly MJ, Brewster SJ, Matise TC, Barrett J, Shea‑Drinkwater M,
et al. A genome‑wide scan for linkage to human exceptional lon‑
gevity identifies a locus on chromosome 4. Proc Natl Acad Sci USA.
2001;98:10505–8.
20. Boyden SE, Kunkel LM. High‑density genomewide linkage analysis of
exceptional human longevity identifies multiple novel loci. PLoS ONE.
2010;5:e12432.
21. Chen YF, Wu CY, Kirby R, Kao CH, Tsai TF. A role for the CISD2 gene in
lifespan control and human disease. Ann N Y Acad Sci. 2010;1201:58–64.
22. Huang YL, Shen ZQ, Wu CY, Teng YC, Liao CC, Kao CH, et al. Compara‑
tive proteomic profiling reveals a role for Cisd2 in skeletal muscle aging.
Aging Cell. 2018;17:e12705.
23. Shen ZQ, Chen YF, Chen JR, Jou YS, Wu PC, Kao CH, et al. CISD2 hap‑
loinsufficiency disrupts calcium homeostasis, causes nonalcoholic
fatty liver disease, and promotes hepatocellular carcinoma. Cell Rep.
2017;21:2198–211.
24. Yeh CH, Shen ZQ, Hsiung SY, Wu CY, Wu PC, Teng YC, et al. Cisd2 is essen‑
tial to delaying cardiac aging and to maintaining heart electromechanical
functions. PLoS Biol. 2019;17:e3000508.
25. Conlan AR, Axelrod HL, Cohen AE, Abresch EC, Zuris J, Yee D, et al. Crystal
structure of Miner1: the redox‑active 2Fe‑2S protein causative in Wolfram
Syndrome 2. J Mol Biol. 2009;392:143–53.
26. Ruiz‑Meana M, Minguet M, Bou‑Teen D, Miro‑Casas E, Castans C, Castel‑
lano J, et al. Ryanodine receptor glycation favors mitochondrial damage
in the senescent heart. Circulation. 2019;139:949–64.
27. Szymański J, Janikiewicz J, Michalska B, Patalas‑Krawczyk P, Perrone M,
Ziółkowski W, et al. Interaction of mitochondria with the endoplasmic
reticulum and plasma membrane in calcium homeostasis, lipid trafficking
and mitochondrial structure. Int J Mol Sci. 2017;18:1576.
28. Zheng P, Chen Q, Tian X, Qian N, Chai P, Liu B, et al. DNA damage triggers
tubular endoplasmic reticulum extension to promote apoptosis by
facilitating ER‑mitochondria signaling. Cell Res. 2018;28:833–54.
29. Ghemrawi R, Battaglia‑Hsu S‑F, Arnold C. Endoplasmic reticulum stress in
metabolic disorders. Cells. 2018;7:63.
30. Yeh CH, Chou YJ, Kao CH, Tsai TF. Mitochondria and calcium homeostasis:
Cisd2 as a big player in Cardiac Ageing. Int J Mol Sci. 2020;21:9238.
31. Huang YL, Shen ZQ, Huang CH, Lin CH, Tsai TF. Cisd2 slows down liver
aging and attenuates age‑related metabolic dysfunction in male mice.
Aging Cell. 2021;20:e13523.
32. Chen YF, Chou T Y, Lin IH, Chen CG, Kao CH, Huang GJ, et al. Upregulation
of Cisd2 attenuates Alzheimer’s‑related neuronal loss in mice. J Pathol.
2020;250:299–311.
33. Shen ZQ, Huang YL, Teng YC, Wang TW, Kao CH, Yeh CH, et al. CISD2
maintains cellular homeostasis. Biochim Biophys Acta Mol Cell Res.
2021;1868:118954.
34. Lee EC, Yu D, De Velasco JM, Tessarollo L, Swing DA, Court DL, Jenkins
NA, Copeland NG. A highly efficient Escherichia coli‑based chromosome
engineering system adapted for recombinogenic targeting and subclon‑
ing of BAC DNA. Genomics. 2001;73:56–65.
35. Lee KH, Morris‑Natschke S, Qian K, Dong Y, Yang X, Zhou T, Belding E, et al.
Recent progress of research on herbal products used in traditional chi‑
nese medicine: the Herbs belonging to The Divine Husbandman’s Herbal
Foundation Canon (Shén Nóng Běn Cǎo Jīng). J Tradit Complement Med.
2012;2(1):6–26.
36. Breton RC, Reynolds WF. Using NMR to identify and characterize natural
products. Nat Prod Rep. 2013;30(4):501–24.
37. Fukushi E. Advanced NMR approaches for a detailed structure analysis of
natural products. Biosci Biotechnol Biochem. 2006;70(8):1803–12.
38. Teng YC, Wang JY, Chi YH, Tsai TF. Exercise and the Cisd2 prolongevity
gene: two promising strategies to delay the aging of skeletal muscle. Int J
Mol Sci. 2020;21:9059.
39. Ward SR, Lieber RL. Density and hydration of fresh and fixed human
skeletal muscle. J Biomech. 2005;38:2317–20.
40. Wang PY, Koishi K, McGeachie AB, Kimber M, Maclaughlin DT, Donahoe
PK, et al. Mullerian inhibiting substance acts as a motor neuron survival
factor in vitro. Proc Natl Acad Sci USA. 2005;102:16421–5.
41. Casaclang‑Verzosa G, Enriquez‑Sarano M, Villaraga HR, Miller JD.
Echocardiographic approaches and protocols for comprehensive
phenotypic characterization of valvular heart disease in mice. J Vis Exp.
2017;120:54110.
42. Nair AB, Jacob S. A simple practice guide for dose conversion between
animals and human. J Basic Clin Pharm. 2016;7:27–31.
43. Lopez‑Otin C, Galluzzi L, Freije JMP, Madeo F, Kroemer G. Metabolic con‑
trol of longevity. Cell. 2016;166:802–21.
44. Distefano G, Goodpaster BH. Effects of exercise and aging on skeletal
muscle. Cold Spring Harb Perspect Med. 2018;8:a029785.
45. Chia CW, Egan JM, Ferrucci L. Age‑related changes in glucose metabo‑
lism, hyperglycemia, and cardiovascular Risk. Circ Res. 2018;123:886–904.
46. Petersen MC, Shulman GI. Mechanisms of insulin action and insulin resist‑
ance. Physiol Rev. 2018;98:2133–223.
47. Santoleri D, Titchenell PM. Resolving the paradox of hepatic insulin resist‑
ance. Cell Mol Gastroenterol Hepatol. 2019;7:447–56.
48. Kim DH, Bang E, Ha S, Jung HJ, Choi YJ, Yu BP, Chung HY. Organ‑differen‑
tial roles of Akt/FoxOs axis as a key metabolic modulator during aging.
Aging Dis. 2021;12:1713–28.
49. Khan RS, Bril F, Cusi K, Newsome PN. Modulation of insulin resistance in
nonalcoholic fatty liver disease. Hepatology. 2019;70:711–24.
50. Nakamura S, Tsutou A, Mizuta K, Negami A, Nakaza T, Hashimoto E,
Yamamura H. Calcium‑calmodulin‑dependent activation of porcine liver
phosphorylase kinase. FEBS Lett. 1983;159:47–50.
51. Agius L. Role of glycogen phosphorylase in liver glycogen metabolism.
Mol Asp Med. 2015;46:34–45.
52. Gupta G, Cases JA, She L, Ma XH, Yang XM, Hu M, et al. Ability of insulin
to modulate hepatic glucose production in aging rats is impaired by fat
accumulation. Am J Physiol Endocrinol Metab. 2000;278:E985–91.
53. Johnson ML, Robinson MM, Nair KS. Skeletal muscle aging and the mito‑
chondrion. Trends Endocrinol Metab. 2013;24:247–56.
54. Eisenberg T, Abdellatif M, Schroeder S, Primessnig U, Stekovic S, Pendl T,
et al. Cardioprotection and lifespan extension by the natural polyamine
spermidine. Nat Med. 2016;22:1428–38.
55. Biernacka A, Frangogiannis NG. Aging and cardiac fibrosis. Aging Dis.
2011;2:158–73.
56. Conrad N, Judge A, Tran J, Mohseni H, Hedgecott D, Crespillo AP, et al.
Temporal trends and patterns in heart failure incidence: a population‑
based study of 4 million individuals. Lancet. 2018;391:572–80.
57. Lazzarini V, Mentz RJ, Fiuzat M, Metra M, O’Connor CM. Heart failure in
elderly patients: distinctive features and unresolved issues. Eur J Heart
Fail. 2013;15:717–23.
58. Guallar E, Stranges S, Mulrow C, Appel LJ, Miller ER 3rd. Enough is
enough: stop wasting money on vitamin and mineral supplements. Ann
Intern Med. 2013;159:850–1.
59. Sadowska‑Bartosz I, Bartosz G. Effect of antioxidants supplementation on
aging and longevity. BioMed Res Int. 2014;2014:404680.
60. Matsumoto H, Ikoma Y, Sugiura M, Yano M, Hasegawa Y. Identification and
quantification of the conjugated metabolites derived from orally admin‑
istered hesperidin in rat plasma. J Agric Food Chem. 2004;52:6653–9.
61. Yamamoto M, Jokura H, Hashizume K, Ominami H, Shibuya Y, Suzuki
A, et al. Hesperidin metabolite hesperetin‑7‑O‑glucuronide, but not
Page 21 of 21
Yehetal. Journal of Biomedical Science (2022) 29:53
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hesperetin‑3’‑O‑glucuronide, exerts hypotensive, vasodilatory, and anti‑
inflammatory activities. Food Funct. 2013;4:1346–51.
62. Choi EJ, Kim GD, Chee KM, Kim GH. Effects of hesperetin on vessel
structure formation in mouse embryonic stem (mES) cells. Nutrition.
2006;22:947–51.
63. Kim JY, Jung KJ, Choi JS, Chung HY. Hesperetin: a potent antioxidant
against peroxynitrite. Free Radic Res. 2004;38:761–9.
64. Roohbakhsh A, Parhiz H, Soltani F, Rezaee R, Iranshahi M. Molecular
mechanisms behind the biological effects of hesperidin and hespere‑
tin for the prevention of cancer and cardiovascular diseases. Life Sci.
2015;124:64–74.
65. Maiti K, Mukherjee K, Murugan V, Saha BP, Mukherjee PK. Exploring the
effect of Hesperetin‑HSPC complex—a novel drug delivery system on
the in vitro release, therapeutic efficacy and pharmacokinetics. AAPS
PharmSciTech. 2009;10:943–50.
66. Deng W, Jiang D, Fang Y, Zhou H, Cheng Z, Lin Y, et al. Hesperetin protects
against cardiac remodelling induced by pressure overload in mice. J Mol
Histol. 2013;44:575–85.
67. Wang B, Li L, Jin P, Li M, Li J. Hesperetin protects against inflamma‑
tory response and cardiac fibrosis in postmyocardial infarction mice
by inhibiting nuclear factor kappaB signaling pathway. Exp Ther Med.
2017;14:2255–60.
68. Kim HJ, Jeon SM, Lee MK, Cho YY, Kwon EY, Lee JH, et al. Comparison of
hesperetin and its metabolites for cholesterol‑lowering and antioxidative
efficacy in hypercholesterolemic hamsters. J Med Food. 2010;13:808–14.
69. Smith DLJ, Nagy TR, Allison DB. Calorie restriction: what recent
results suggest for the future of ageing research. Eur J Clin Invest.
2010;40:440–50.
70. Duggal NA. Reversing the immune ageing clock: lifestyle modifications
and pharmacological interventions. Biogerontology. 2018;19:481–96.
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Non‐alcoholic fatty liver disease (NAFLD) has an estimated prevalence of 25% in the general population, and cirrhosis secondary to Non‐Alcoholic Steatohepatitis (NASH) is predicted to become the leading cause of liver transplantation and yet there is a lack of effective licensed treatments. There is a close relationship between insulin resistance and NAFLD, with the prevalence of NAFLD being five‐fold higher in patients with diabetes compared to patients without. Insulin resistance is implicated both in the pathogenesis of NAFLD and in disease progression from steatosis to NASH. Thus modulation of insulin resistance represents a potential strategy for NAFLD treatment. This review highlights key proposed mechanisms linking insulin resistance and NAFLD, such as changes in rates of adipose tissue lipolysis and de novo lipogenesis, impaired mitochondrial fatty acid oxidation, changes in fat distribution, alterations in the gut microbiome, and alterations in levels of adipokines and cytokines. Further, it goes on to discuss the main pharmacological strategies used to treat insulin resistance in patients with NAFLD, and their efficacy based on recently published experimental and clinical data. These include (biguanides, glucagon‐like peptide 1 receptor (GLP‐1) agonists, Dipeptidyl peptidase 4 (DPP‐4) inhibitors, Peroxisome Proliferator‐Activated Receptor (PPAR‐ γ/ α/δ) agonists, Sodium Glucose Cotransporter (SGLT2) inhibitors and Farnesoid X receptor (FXR) agonists), with further novel treatments on the horizon. Ideally treatment would improve insulin resistance, reduce cardiovascular risk and produce demonstrable improvements in NASH histology ‐ this is likely to be achieved with a combinatorial approach. This article is protected by copyright. All rights reserved.
Article
Background: Senescent cardiomyocytes exhibit a mismatch between energy demand and supply that facilitates their transition toward failing cells. Altered calcium transfer from sarcoplasmic reticulum (SR) to mitochondria has been causally linked to the pathophysiology of aging and heart failure. Methods: Because advanced glycation-end products accumulate throughout life, we investigated whether intracellular glycation occurs in aged cardiomyocytes and its impact on SR and mitochondria. Results: Quantitative proteomics, Western blot and immunofluorescence demonstrated a significant increase in advanced glycation-end product-modified proteins in the myocardium of old mice (≥20months) compared with young ones (4-6months). Glyoxalase-1 activity (responsible for detoxification of dicarbonyl intermediates) and its cofactor glutathione were decreased in aged hearts. Immunolabeling and proximity ligation assay identified the ryanodine receptor (RyR2) in the SR as prominent target of glycation in aged mice, and the sites of glycation were characterized by quantitative mass spectrometry. RyR2 glycation was associated with more pronounced calcium leak, determined by confocal microscopy in cardiomyocytes and SR vesicles. Interfibrillar mitochondria-directly exposed to SR calcium release-from aged mice had increased calcium content compared with those from young ones. Higher levels of advanced glycation-end products and reduced glyoxalase-1 activity and glutathione were also present in atrial appendages from surgical patients ≥75 years as compared with the younger ones. Elderly patients also exhibited RyR2 hyperglycation and increased mitochondrial calcium content that was associated with reduced myocardial aerobic capacity (mitochondrial O2 consumption/g) attributable to less respiring mitochondria. In contracting HL-1 cardiomyocytes, pharmacological glyoxalase-1 inhibition recapitulated RyR2 glycation and defective SR-mitochondria calcium exchange of aging. Conclusions: Mitochondria from aging hearts develop calcium overload secondary to SR calcium leak. Glycative damage of RyR2, favored by deficient dicarbonyl detoxification capacity, contributes to calcium leak and mitochondrial damage in the senescent myocardium.