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Glucosamine Extends the Lifespan of Caenorhabditis elegans via Autophagy Induction

Authors:
  • The Japanese Clinical Nutrition Association

Abstract

Glucosamine (GlcN) is commonly used as a dietary supplement to promote cartilage health in humans. We previously reported that GlcN could induce autophagy in cultured mammalian cells. Autophagy is known to be involved in the prevention of various diseases and aging. Here, we showed that GlcN extended the lifespan of the nematode Caenorhabditis elegans by inducing autophagy. Autophagy induction by GlcN was demonstrated by western blotting for LGG-1 (an ortholog of mammalian LC3) and by detecting autophagosomal dots in seam cells by fluorescence microscopy. Lifespan assays revealed that GlcN-induced lifespan extension was achieved with at least 5 mM GlcN. A maximum lifespan extension of approximately 30 % was achieved with 20 mM GlcN (p<0.0001). GlcN-induced lifespan extension was not dependent on the longevity genes daf-16 and sir-2.1 but dependent on the autophagy-essential gene atg-18. Therefore, we suggest that oral administration of GlcN could help delay the aging process via autophagy induction.
Regular Paper
Glucosamine Extends the Lifespan of Caenorhabditis elegans
via Autophagy Induction
Glucosamine Extends Nematode Lifespan via Autophagy Induction
(Received April 3, 2018; Accepted May 24, 2018)
(J-STAGE Advance Published Date: June 12, 2018)
Tomoya Shintani,1,2, Yuhei Kosuge,1, and Hisashi Ashida3, †
1Graduate School of Biostudies, Kyoto University
(Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606–8502, Japan)
2United Graduate School of Agriculture, Ehime University
(3–5–7, Tarumi, Matsuyama, Ehime 790–8507, Japan)
3Faculty of Biology-Oriented Science and Technology, Kindai University
(930 Nishimitani, Kinokawa, Wakayama 649–6493, Japan)
Abstract: Glucosamine (GlcN) is commonly used as a dietary supplement to promote cartilage health in
humans. We previously reported that GlcN could induce autophagy in cultured mammalian cells.
Autophagy is known to be involved in the prevention of various diseases and aging. Here, we showed
that GlcN extended the lifespan of the nematode Caenorhabditis elegans by inducing autophagy.
Autophagy induction by GlcN was demonstrated by western blotting for LGG-1 (an ortholog of
mammalian LC3) and by detecting autophagosomal dots in seam cells by fluorescence microscopy.
Lifespan assays revealed that GlcN-induced lifespan extension was achieved with at least 5 mM GlcN. A
maximum lifespan extension of approximately 30 % was achieved with 20 mM GlcN (p<0.0001). GlcN-
induced lifespan extension was not dependent on the longevity genes daf-16 and sir-2.1 but dependent
on the autophagy-essential gene atg-18. Therefore, we suggest that oral administration of GlcN could
help delay the aging process via autophagy induction.
Key words: anti-aging, atg-18, autophagy, Caenorhabditis elegans, glucosamine, lifespan
INTRODUCTION
Glucosamine (2-amino-2-deoxy-D-glucose, GlcN) is the
constitutional unit of chitosan and chitin, which are pro‐
duced in nature by arthropods, fungi, and cephalopods.
GlcN is industrially manufactured for dietary supplements
by the hydrolysis of crustacean exoskeletons, which are
mainly composed of chitin. GlcN has been reported to ef‐
fectively prevent and treat osteoarthritis in humans1)2) and
have a positive effect on skin aging in a clinical study.3) On
the other hand, a meta-analysis has reported the ineffective‐
ness of GlcN on articulation.4) Therefore, the anti-osteoar‐
thritis effect of GlcN remains controversial. Thus far, many
people have taken GlcN as a dietary supplement. Accord‐
ing to a large-scale epidemiological study on consumers of
various dietary supplements, use of GlcN was reported to
associate with decreased total mortility.5) However, the un‐
derlying molecular mechanism of the longevity effect of
GlcN remains unclear.
Autophagy is a cellular process that nonspecifically de‐
grades cytosolic components. An autophagic membrane en‐
Corresponding author (Tel. +81–736–77–3888, Fax. +81–736–77–
4754, E-mail: ashida@waka.kindai.ac.jp).
Tomoya Shintani and Yuhei Kosuge contributed equally to this work.
Abbreviations: FUdR, 5-fluoro-2′-deoxyuridine; GFP, green fluorescent
protein; GPI, glycosylphosphatidylinositol; LC3, microtubule-associ‐
ated protein 1 light chain 3; mTOR, mammalian target of rapamycin;
NGM, nematode growth medium; PE, phosphatidylethanolamine.
gulfs parts of the cytosol containing proteins and organelles
and is fused with the lysosome to degrade inner compo‐
nents.6) Autophagy is known to play critical roles in various
cellular processes such as the response to starvation,7) the
prevention of bacterial infection,8)9) antigen presentation,10)
neural development,11) glycogen degradation,12) and lipid
metabolism.13) In contrast to autophagy induced in response
to environmental signals, basal level autophagy may also
have important functions, which include protein quality
control and cellular anti-aging functions.14)15) Therefore, the
induction of autophagy by drugs has attracted attention as a
promising anti-aging approach.16) Several autophagy induc‐
ers have been reported to ameliorate the toxicity of poly‐
glutamine-expanded huntingtin and related proteinopa‐
thies16) and extend the lifespan of animal models.17)
Caenorhabditis elegans is a common animal model for
lifespan assays. The lifespan of C. elegans is known to be
mediated by several proteins.18) DAF-16, an ortholog of
mammalian FOXO transcription factors, is regulated by
TOR signaling and can affect lifespan.19) In addition,
SIR-2.1 (NAD+-dependent protein deacetylase), an ortho‐
log of mammalian SIRT1, is a longevity-related protein.20)
A study found that resveratrol could extend the lifespan of
nematodes and fruit flies in a SIR-2.1/Sir2-dependent man‐
ner without reducing fertility.21) Aging could also be medi‐
ated by autophagy and autophagy-related genes.22)
Previously, we reported that GlcN supplementation with‐
in a physiological concentration range could strongly in‐
J. Appl. Glycosci., 65, 37–43 (2018)
doi: 10.5458/jag.jag.JAG-2018_002
©2018 The Japanese Society of Applied Glycoscience
duce autophagy in mammalian cells.23) To investigate the
autophagy-inducing effect of GlcN on aging, we selected
the nematode C. elegans as a model. C. elegans is com‐
monly used as an aging model and for autophagy
studies.22)24) Here, we demonstrated the autophagy-inducing
activity of GlcN in the nematode. In addition, at least 5 mM
GlcN extended the lifespan of the nematode in an autopha‐
gy-dependent manner.
MATERIALS AND METHODS
Chemicals. GlcN hydrochloride was purchased from Naca‐
lai Tesque, INC. (Kyoto, Japan). Other chemicals (not men‐
tioned in the following sections) were obtained from Wako
Pure Chemical Industries (Osaka, Japan).
Strains. The C. elegans strains N2 (wild type), DA2123
(asIs2122[lgg-1::GFP + rol(su1006)], and atg-18(gk378)
were provided by the Caenorhabditis Genetic Center
(CGC). The strains daf-16(mgDf50) and sir-2.1(ok434)
were provided from Prof. Eisuke Nishida (Kyoto Universi‐
ty, Kyoto, Japan).25) Escherichia coli OP50 was used as a
food source for the nematodes.26)
C. elegans culture and preparation of L1 larvae. C. ele‐
gans was maintained at 20 °C on nematode growth medium
(NGM) (2.0 % agar, 0.5 % peptone, 50 mM NaCl, 25 mM
potassium phosphate buffer pH 6.0, 1 mM CaCl2, 1 mM
MgSO4, and 5 µg/mL cholesterol) with E. coli OP50 as
food source.26) The eggs of C. elegans were collected by
treating egg-bearing adult worms with alkaline hypochlor‐
ite solution, and they were shaken in S basal medium at
20 °C for 20–24 h to prepare synchronized first-stage lar‐
vae (L1).27)
Western blotting. Worms were lysed in 20 mM Tris/HCl
buffer (pH 7.4) containing 1.0 % Triton X-100, 150 mM
NaCl, and cOmplete™ Protease Inhibitor Cocktail (F. Hoff‐
mann-La Roche AG, Basel, Switzerland). After centrifuga‐
tion at 12,000 rpm for 10 min, the supernatants were ob‐
tained. Protein concentration was measured using BCA
Protein Assay Kit (Thermo Fisher Scientific, Inc., Wal‐
tham, MA, USA). Supernatants containing 20 µg of pro‐
teins were separated by a 15 % SDS-PAGE gel under re‐
ducing conditions and blotted onto a PVDF membrane. The
membrane was blocked with 5.0 % bovine serum albumin
(Nacalai Tesque) for the detection of phosphorylated pro‐
teins and 1.0 % skim milk (Wako Pure Chemical Indus‐
tries) for other proteins. The primary antibody was mouse
anti-GFP IgG (1/1,000; Sigma-Aldrich Corporation, St.
Louis, MO, USA). The secondary antibody was horseradish
peroxidase-conjugated anti-mouse IgG (1/4,000; MEDI‐
CAL & BIOLOGICAL LABORATORIES CO., LTD., Na‐
goya, Japan). Detection of the target proteins was carried
out using West Pico Chemiluminescent Kit (Thermo Fisher
Scientific) and LAS Image Analyzer (Fuji Film Corpora‐
tion, Tokyo, Japan).
Fluorescence microscopy. One drop of 10 mM sodium
azide (in M9 buffer; 6.0 g/L Na2HPO4, 3.0 g/L KH2PO4, 5.0
g/L NaCl, 0.25 g/L MgSO47H2O) was placed onto a glass
slide followed by a drop of M9 buffer containing nemato‐
des, and a cover glass was placed on top. The transgenic
worms expressing GFP::LGG-128) on the glass slide were
analyzed using a fluorescence microscope (Olympus IX-70;
Olympus Corporation, Tokyo, Japan). The GFP dots of ap‐
proximately 100 seam cells were counted for each worm
group.
Lifespan assay. Lifespan assays were carried out at
20 °C.27) L1 nematodes were transferred to culture plates
containing NGM with E. coli OP50. 5-Fluoro-2′-deoxyuri‐
dine (FUdR) was added (50 µM, final concentration) to the
NGM plates to prevent progeny growth. Then, 50
synchronized animals (10 or 25 animals/plate) were placed
on NGM plates containing GlcN with UV-killed E. coli
OP50. Control worms were incubated in medium without
GlcN. In each assay, 2 plates (50 animals) were used for the
same condition. The numbers of live and dead animals
were counted every second day under a microscope based
on their movement. Each assay was repeated twice except
for the assays using N2 adults (30 mM GlcN), atg-18 mu‐
tants, and sir-2.1 mutants. The survival curves were deter‐
mined using the Kaplan-Meier method, and survival differ‐
ences were tested for significance using the log-rank test.
Data are expressed as the mean ± standard error (SE).
RESULTS
Autophagy induction in C. elegans by GlcN.
In a previous study, we found that GlcN could strongly
induce autophagy in mammalian cells such as HeLa and
COS cells.23) For the detection of autophagy induction, mi‐
crotubule-associated protein 1 light chain 3 (LC3) is one of
the most suitable marker proteins in mammalian cells.29)
When autophagy is induced, cytosolic LC3-I is covalently
attached to phosphatidylethanolamine (PE) on the autopha‐
gosomal membrane to form LC3-II. Membrane-anchored
LC3-II is involved in autophagosomal membrane elonga‐
tion, and those on the inner membrane will be degraded in
autolysosomes. To assess the autophagy-inducing activity
of GlcN in C. elegans, we used transgenic worms express‐
ing LGG-1 (an ortholog of mammalian LC3) fused with
GFP. The preliminary results revealed that growth inhibi‐
tion was observed by microscopic analysis when GlcN was
more than 80 mM (data not shown). Therefore,
GFP::LGG-1 worms were grown with 0–80 mM GlcN for
96 h and harvested, and the lysates were analyzed by west‐
ern blotting using anti-GFP antibody (Fig. 1A). PE-conju‐
gated GFP::LGG-1, the autophagosomal membrane-anch‐
ored form, was hardly detected by SDS-PAGE because of
the small difference in migration between PE-conjugated
and -unconjugated GFP::LGG-1. However, the accumula‐
tion of protease-resistant free GFP could be detected, which
might be transferred into autolysosomes via autophagy acti‐
vation and released from PE-conjugated GFP::LGG-1 by
lysosomal protease. Therefore, the level of GFP would cor‐
respond to the degree of autophagy induction. In this assay,
we detected free GFP with 40–80 mM GlcN but not 20 mM
GlcN. Next, autophagosome formation was analyzed by
fluorescence microscopy using the same transgenic ani‐
mals, which were continuously cultured in the presence of
40 mM GlcN. GFP-positive dots were observed in both the
38 J. Appl. Glycosci., Vol. 65, No. 3 (2018)
GlcN-treated eggs/embryos and adults but not in the control
worms (Fig. 1B). In adult worms, seam cells arranged as
longitudinal rows on the left and right sides of the body
were used for counting autophagosomes.30) Approximately
100 cells were analyzed by fluorescence microscopy. A sig‐
nificant increase (p<0.05) in the number of GFP-positive
dots was observed in the cytosol of the seam cells of worms
grown with 5–80 mM GlcN (Fig. 1C). A similar pattern
was observed with 40 mM GlcN; however, it was not sig‐
nificant. Taken together, these results suggest that 5–80
mM GlcN significantly induced autophagy activation. In‐
consistency in autophagy induction levels and required
GlcN concentration in Figs. 1A–1C might be due to differ‐
ences in cell types and detection methods.
 
Lifespan extension of C. elegans by GlcN.
To assess the effect of GlcN on the lifespan of nemato‐
des, synchronized young adults of the wild-type strain were
treated with FUdR to prevent oviposition. They were
grown in the presence of 0–30 mM GlcN, and their lifespan
Autophagy induction by GlcN in C. elegans.
 (A) Transgenic worms expressing GFP::LGG-1 were grown with 0–80 mM GlcN for 96 h and harvested. GFP::LGG-1 and free GFP were
detected by western blotting using anti-GFP antibody. (B) Autophagosome formation was analyzed by fluorescence microscopy. Left column,
control; right column, worms grown in medium containing 40 mM GlcN. Upper row, Nomarski images of eggs/embryos; middle row, fluorescent
images of eggs/embryos; bottom row, fluorescent images of adults. (C) The number of GFP-positive dots in the cytosol of seam cells was deter‐
mined. Approximately 100 seam cells were analyzed. Data represent the mean ± standard deviation. Asterisks indicate significant differences
compared with the control (p<0.05) by Student's t-test.
Fig. 1.
Survival curves of C. elegans grown with GlcN.
 (A) Young adults of the wild-type strain (N2) were grown with GlcN (0–30 mM). (B) Larvae of the wild-type strain (N2) were grown with
GlcN (0–30 mM). (C) The daf-16 mutant worms were grown with or without 10 mM GlcN. (D) The sir-2.1 mutant worms were grown with or
without 10 mM GlcN. (E) The atg-18 mutant worms were grown with or without 10–20 mM GlcN.
Fig. 2.
Shintani et al.: Glucosamine Extends Nematode Lifespan via Autophagy Induction 39
was measured (Fig. 2A). In the presence of 5–20 mM
GlcN, the mean lifespan was significantly increased
(p<0.01) compared with that of the control. The same ex‐
periments were independently performed twice, and the re‐
sults are shown in Table 1. In these experiments, 15 mM
GlcN was the most effective, demonstrating a lifespan ex‐
tension of 22 %. A higher GlcN concentration (25–30 mM)
was less effective for lifespan extension. In addition, we in‐
vestigated the effect of GlcN on the total lifespan from the
larval stage. Eggs were hatched in GlcN-containing medi‐
um. Then, the larvae were grown on the same plate, and
their lifespan was measured. In this assay, 10–25 mM GlcN
significantly increased the lifespan (p<0.0001), and a maxi‐
mum lifespan extension of around 30 % was achieved with
20–25 mM GlcN (Fig. 2B, Table 1). Overall, 10–20 mM
GlcN was found to be the most effective for the lifespan
extension of C. elegans.
 
Lifespan extension of C. elegans via an autophagy-de‐
pendent mechanism.
Thus far, several molecules involved in longevity, such
as DAF-16 and SIR-2.1, have been identified in
C. elegans.18) The former is the sole ortholog of the FOXO
transcription factors of mammals, and the latter is an
NAD+-dependent histone deacetylase. To determine wheth‐
er the lifespan extension effect of GlcN is mediated via
pathways involving these molecules, we used daf-16 and
sir-2.1 mutants. These mutants were grown from eggs with
or without 10 mM GlcN. The lifespan of both daf-16 and
sir-2.1 mutants was significantly increased (p<0.0001) in
the presence of GlcN (Figs. 2C and 2D, Table 1). These re‐
sults indicated that GlcN-induced lifespan extension was
independent of DAF-16 and SIR-2.1. Next, we investigated
whether lifespan extension is induced by GlcN in an au‐
tophagy-dependent manner. The autophagy-defective
atg-18 mutant has been reported to be short-lived, and it
does not accumulate LGG-1 at the embryonic stage.31) The
lifespan of the atg-18 mutant was not affected by 10–20
mM GlcN (Fig. 2E, Table 1). Taken together, these results
clearly indicated that GlcN-induced lifespan extension was
dependent on autophagy but not dependent on common
longevity pathways that involve DAF-16 and SIR-2.1.
DISCUSSION
Thus far, the most common strategy for promoting lon‐
gevity is calorie restriction. An appropriate level of calorie
restriction has been experimentally demonstrated to elon‐
gate the lifespan of various animal models such as rhesus
monkeys, mice, fruit flies, and nematodes.32) On the other
hand, the intake of certain exogenous factors has also been
reported to induce longevity.33) These longevity-inducing
factors include resveratrol, rapamycin, spermidine, and 2-
deoxy-glucose. Most of these factors and calorie restriction
have been found to induce autophagy; thus, autophagy has
been recognized as one of the most important cellular
mechanisms that prevent aging.34)35) Resveratrol, a polyphe‐
nol found in red wine, can induce autophagy by activating
sirtuin, an NAD+-dependent histone deacetylase.32) Rapa‐
mycin, an antibiotic produced by actinomycete, can directly
inhibit TOR, which negatively regulates autophagy.32) Sper‐
midine, a type of polyamine, can also induce autophagy
through histone acetyltransferase inhibition in a dependent
or independent manner.36) 2-Deoxy-glucose is a well known
Summary and statistical analysis of C. elegans lifespan assays.
Strain GlcN
(mM)
Mean lifespan
± SE (days)
Extension
(%)
Number of
worms
p-value
(vs. control)
N2 (starting from adult) 0 19.0 ± 1.5 100
5 22.1 ± 1.8 16 100 <0.001
10 22.3 ± 0.1 17 100 <0.0001
15 23.2 ± 2.1 22 100 <0.001
20 21.2 ± 1.6 12 100 <0.01
25 18.4 ± 0.1 –3 50 NS
30 20.1 ± 0.1 6 50 <0.01
N2 (starting from egg) 0 18.2 ± 0.5 100
5 19.7 ± 1.0 8 100 NS
10 22.0 ± 0.9 21 100 <0.0001
15 21.8 ± 0.1 20 100 <0.0001
20 23.8 ± 0.1 31 100 <0.0001
25 23.7 ± 1.0 30 100 <0.0001
30 20.3 ± 0.1 12 100 <0.01
daf-16(mgDf50) 0 15.5 ± 0.3 100
10 18.9 ± 1.0 22 100 <0.0001
sir-2.1(ok434) 0 18.4 ± 0.1 50
10 24.4 ± 0.1 33 50 <0.0001
atg-18(gk378) 0 15.9 ± 0.1 50
10 15.2 ± 0.1 –4 50 NS
20 16.0 ± 0.1 1 50 NS
The p-value (vs. control) was calculated by the log-rank test. NS, not significant.
Table 1.
40 J. Appl. Glycosci., Vol. 65, No. 3 (2018)
inhibitor of glycolysis that can deplete cellular ATP and act
as a calorie restriction mimetic to induce autophagy.37)38)
We previously reported that GlcN could induce autopha‐
gy in mammalian cells via an mTOR-independent signaling
pathway.23) In this study, we demonstrated for the first time
that GlcN induced autophagy in the adults and embryos of
C. elegans. We also found that GlcN supplementation ex‐
tended the lifespan of the nematode. Recently, another
group reported the longevity effect of GlcN on nematodes
and mice.39) They suggest that the effect was caused by im‐
paired glucose metabolism; however, the involvement of
autophagy was not discussed. Here, we clearly showed that
the longevity effect of GlcN required an autophagy gene,
atg-18. However, unlike functional food factors and calorie
restriction, the longevity genes sir-2.1 and daf-16 were not
required for GlcN-induced lifespan extension.
GlcN is known to be incorporated into cells via glucose
transporters and metabolized to UDP-GlcNAc through
GlcN-6-phosphate.40) UDP-GlcNAc is used for the biosyn‐
thesis of O-linked GlcNAc, N-glycan, and GPI-anchor.
Among these, O-linked GlcNAc is important modification
of intracellular proteins required for regulating insulin sig‐
naling. Therefore, a high concentration of intracellular
UDP-GlcNAc may induce autophagy. The increased syn‐
thesis of N-glycan precursors can also improve protein ho‐
meostasis and extend the lifespan of C. elegans.41)
An excess amount of GlcN-6-phosphate is metabolized
to fructose-6-phosphate (an intermediate of glycolysis) and
ammonia by glucosamine-6-phosphate deaminase. Intracel‐
lular ammonia has been reported to induce autophagy via
UNC-51-like kinase 1 (ULK1)/ULK2-independent path‐
ways.42) We found that mammalian cells cultured with am‐
monia induced autophagy in an mTOR-independent man‐
ner (unpublished data). However further studies would be
needed to elucidate the autophagy-inducing mechanism of
GlcN in terms of UDP-GlcNAc and/or ammonia.
Recently, non-metabolizable D-allulose, one of the rare
hexoses, has been reported to extend the lifespan of nemat‐
odes.43) Similar to GlcN and 2-deoxy-glucose, D-allulose
enters into cells through glucose transporters and inhibits
glycolysis, inducing the metabolism of stored fat and mito‐
chondrial respiration via AMP-activated protein kinase
(AMPK). Increased respiration can cause the temporary
formation of reactive oxygen species, leading to increased
anti-oxidative enzyme activity, oxidative stress resistance,
and survival rates.43)44) Orally administrated GlcN has also
been reported to affect carbohydrate metabolism and reduce
body fat in rodents,45) and it could contribute to enhanced
oxidative stress resistance followed by AMPK activation.39)
Therefore, the mechanism of the anti-aging effect of GlcN
may be partially similar to that of D-allulose and 2-deoxy-
glucose.
GlcN is widely consumed as a dietary supplement to pro‐
mote cartilage health; however, its efficacy remains contro‐
versial. A large-scale epidemiological study on the long-
term intake of various dietary supplements has revealed
that the use of GlcN or chondroitin could significantly re‐
duce mortality.5) Despite its limitations, the results of this
study were consistent, to some extent, with those of the epi‐
demiological study. Therefore, GlcN may exert anti-aging
effects by inducing autophagy in humans.
ACKNOWLEDGMENTS
We thank Prof. Eisuke Nishida (Kyoto University, Kyoto, Ja‐
pan) for the valuable advice and for providing the
daf-16(mgDf50) and sir-2.1(ok434) strains. Furthermore, we
thank Prof. Masashi Sato (Kagawa University, Kagawa, Japan)
for the helpful discussion. This study was supported by JSPS
KAKENHI grants 24580179 and 15K07448 (to H.A.). This
study was also partially supported by the Sasakawa Scientific
Research Grant from The Japan Science Society (to T.S.).
The authors declare no conflicts of interest. However,
Tomoya Shintani is an employee of Matsutani Chemical In‐
dustry Co., Ltd. (Hyogo, Japan).
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... [22,23]. In addition, two animal experiments in 2014 and 2018 showed that the lifespan of mice and nematodes was extended with orally administered glucosamine [24,25]. In the 2014 mouse experiment, experimental data showed that the average lifespan of mice was extended by approximately 10% with improving energy metabolism. ...
... Although it may be involved in the regulation of sugar and amino acid metabolism as described above, the details of the anti-aging mechanisms of glucosamine are still unknown, with the exception of its efficacy in carbohydrate metabolism modulation [24] and autophagy induction [25,38]. ...
... However, the company provided no financial support for this study. This graph is based on data from our reference [25]. Low dose refers to 10 mM and high dose refers to 25 mM as the glucosamine concentration in the nematode feed. ...
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d-Glucosamine is a protective dietary supplement or medicine for osteoarthritis of the knee, a musculoskeletal disease that leads to a significant deterioration in daily activities and quality of life. As glucosamine can restore damaged cartilage worn down by joint disease, there was hope it could also improve symptoms. Data from several clinical studies on the efficacy of glucosamine on knee joint function conducted since the 1980s have been used in certain meta-analyses and epidemiological studies since 2010, yet the effect of glucosamine on the knee joints remains controversial. Concurrently, many drugs have been investigated for their anti-aging properties. Among these drugs, d-glucosamine has recently been discovered to be a potential substance with convincing evidence for increasing human lifespan. More interestingly, Zhi-Hao et al. have recently reported that the use of glucosamine was associated with a reduction in total mortality regardless of its effect on the knee (Annals of the Rheumatic Diseases 79(2020)829–836). Additionally, glucosamine prolongs the lifespan of the nematode Caenorhabditis elegans, possibly due to its calorie restriction-mimicking effect by improving energy metabolism and inducing autophagy. Thus, the recent large-scale epidemiological report on glucosamine intake and mortality, as well as our animal studies, has become relevant. However, the potential significance of O-GlcNAcylation of proteins by glucosamine in anti-aging should be more clearly investigated as another mechanism in the future. This paper presents the novel concept of repositioning glucosamine from a dietary supplement or an OTC drug for osteoarthritis improvements to an anti-aging drug for healthy lifespan extension.
... D-Glucosamine is an amino sugar that serves as a precursor for glycosylated proteins and lipids and acts on glycolysis through hexokinase-1 inhibition. This amino monosaccharide is a CRM candidate due to its lifespan-prolonging effects in nematodes and aging mice (45,82) and its in vivo and in vitro autophagyactivating properties (82)(83)(84). In aging mice it was also shown to induce mitochondrial biogenesis, to lower blood glucose levels (45), and to counteract high-fat diet induced metabolic changes in rats (85), thus mimicking several effects of CR. ...
... D-Glucosamine is an amino sugar that serves as a precursor for glycosylated proteins and lipids and acts on glycolysis through hexokinase-1 inhibition. This amino monosaccharide is a CRM candidate due to its lifespan-prolonging effects in nematodes and aging mice (45,82) and its in vivo and in vitro autophagyactivating properties (82)(83)(84). In aging mice it was also shown to induce mitochondrial biogenesis, to lower blood glucose levels (45), and to counteract high-fat diet induced metabolic changes in rats (85), thus mimicking several effects of CR. ...
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The human diet and dietary patterns are closely linked to the health status. High-calorie Western-style diets have increasingly come under scrutiny as their caloric load and composition contribute to the development of non-communicable diseases, such as diabetes, cancer, obesity, and cardiovascular disorders. On the other hand, calorie-reduced and health-promoting diets have shown promising results in maintaining health and reducing disease burden throughout aging. More recently, pharmacological Caloric Restriction Mimetics (CRMs) have gained interest of the public and scientific community as promising candidates that mimic some of the myriad of effects induced by caloric restriction. Importantly, many of the CRM candidates activate autophagy, prolong life- and healthspan in model organisms and ameliorate diverse disease symptoms without the need to cut calories. Among others, glycolytic inhibitors (e.g., D-allulose, D-glucosamine), hydroxycitric acid, NAD+ precursors, polyamines (e.g., spermidine), polyphenols (e.g., resveratrol, dimethoxychalcones, curcumin, EGCG, quercetin) and salicylic acid qualify as CRM candidates, which are naturally available via foods and beverages. However, it is yet unclear how these bioactive substances contribute to the benefits of healthy diets. In this review, we thus discuss dietary sources, availability and intake levels of dietary CRMs. Finally, since translational research on CRMs has entered the clinical stage, we provide a summary of their effects in clinical trials.
... Page 24 of 36 Liu Natural Products and Bioprospecting (2022) 12:18 and mitochondrial respiration via AMPK. Increased respiration leads to a transient upregulation of ROS production, resulting in increased antioxidant activity, resistance to oxidative stress, and viability [227]. Both d-Alu and GlcN contain a functional hexosaccharide with high safety and health benefits that are thought to extend lifespan. ...
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Human longevity has increased dramatically during the past century. More than 20% of the 9 billion population of the world will exceed the age of 60 in 2050. Since the last three decades, some interventions and many preclinical studies have been found to show slowing aging and increasing the healthy lifespan of organisms from yeast, flies, rodents to nonhuman primates. The interventions are classified into two groups: lifestyle modifications and pharmacological/genetic manipulations. Some genetic pathways have been characterized to have a specific role in controlling aging and lifespan. Thus, all genes in the pathways are potential antiaging targets. Currently, many antiaging compounds target the calorie-restriction mimetic, autophagy induction, and putative enhancement of cell regeneration, epigenetic modulation of gene activity such as inhibition of histone deacetylases and DNA methyltransferases, are under development. It appears evident that the exploration of new targets for these antiaging agents based on biogerontological research provides an incredible opportunity for the healthcare and pharmaceutical industries. The present review focus on the properties of slow aging and healthy life span extension of natural products from various biological resources, endogenous substances, drugs, and synthetic compounds, as well as the mechanisms of targets for antiaging evaluation. These bioactive compounds that could benefit healthy aging and the potential role of life span extension are discussed.
... While acetylation of histone tails is largely ephemeral in nature. Histone methylation is widely observed to be a mark that confers long-standing epigenetic memory [12,13]. This histone modification is accomplished by the catalysis of histone methyltransferase (HMT). ...
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Ageing is characterized by the maintaining deterioration of homeostatic processes over time, leading to functional decline and increased risk of disease and death. Several distinct mechanisms underlying ageing have been reported and mounting shreds of evidence have shown that histone methylation, an epigenetic marker, regulates gene expression during ageing. Recently, SET-domain genes have gained attentions and have been identified as histone methyltransferase involved in ageing process. Deletion of these genes extends lifespan and increased oxida-tive stress resistance in Caenorhabditis elegans depends on the daf-16 activity in the insulin/IGF pathway. In this chapter, we propose to investigate the role of histone methylation in the process of ageing and oxidative stress with an emphasis on the role of set-18 gene in ageing process.
... Glucosamine extends lifespan in mice by inhibiting glycolysis, activating AMPK, and inducing mitochondrial biogenesis (Weimer et al., 2014). In nematodes, glucosamine also extends lifespan by activating autophagy (Shintani et al., 2018). Perhaps more importantly, several epidemiological studies indicate that people who regularly consume glucosamine have lower all-cause mortality (Bell et al., 2012;Pocobelli et al., 2010) and reduced incidence of lung and colon cancer, compared with non-users (Brasky et al., 2011;Kantor et al., 2018), suggesting possible anti-aging effects in humans. ...
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Caloric restriction (CR) mimetics are molecules that produce beneficial effects on health and longevity in model organisms and humans, without the challenges of maintaining a CR diet. Conventional CR mimetics such as metformin, rapamycin and spermidine activate autophagy, leading to recycling of cellular components and improvement of physiological function. We review here novel CR mimetics and anti-aging compounds, such as 4,4 ′-dimethoxychalcone, fungal polysaccharides, inorganic nitrate, and trientine, highlighting their possible molecular targets and mechanisms of action. The activity of these compounds can be understood within the context of hormesis, a biphasic dose response that involves beneficial effects at low or moderate doses and toxic effects at high doses. The concept of hormesis has widespread implications for the identification of CR mimetics in experimental assays, testing in clinical trials, and use in healthy humans. We also discuss the promises and limitations of CR mimetics and anti-aging molecules for delaying aging and treating chronic diseases.
... Given these generally favorable responses, DAlu would appear to have considerable potential as a CRM [69]. Further evidence of this potential was reported in a study by Shintani et al. [82]. They observed increased lifespan in nematodes fed DAlu. ...
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Calorie restriction mimetics encompass a growing research field directed toward developing treatments that mimic the anti-aging effects of long-term calorie restriction without requiring a change in eating habits. A wide range of approaches have been identified that include (1) intestinal inhibitors of fat and carbohydrate metabolism; (2) inhibitors of intracellular glycolysis; (3) stimulators of the AMPK pathway; (4) sirtuin activators; (5) inhibitors of the mTOR pathway, and (6) polyamines. Several biotech companies have been formed to pursue several of these strategies. The objective of this review is to describe the approaches directed toward glycolytic inhibition. This upstream strategy is considered an effective means to invoke a wide range of anti-aging mechanisms induced by CR. Anti-cancer and anti-obesity effects are important considerations in early development efforts. Although many dozens of candidates could be discussed, the compounds selected to be reviewed are the following: 2-deoxyglucose, 3-bromopyruvate, chrysin, genistein, astragalin, resveratrol, glucosamine, mannoheptulose, and d-allulose. Some candidates have been investigated extensively with both positive and negative results, while others are only beginning to be studied.
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Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension in various species. Thus, there is considerable interest in the potential health benefits of dietary supplementation with glucosamine or other HBP metabolites in people. The HBP is a sensor for energy availability and its activation has been implicated in tumor progression and diabetes in higher organisms. As the activation of the HBP has been linked to longevity in lower animals, it is imperative to explore the long-term effects of chronic HBP activation in mammals, which has not been examined so far. To address this issue, we activated the HBP in mice both genetically and through metabolite supplementation, and evaluated metabolism, memory, and survival. GlcNAc supplementation in the drinking water had no adverse effect on weight gain in males but increased weight in young female mice. Glucose or insulin tolerance were not affected up to 20 months of age. Of note, we observed improved memory in the Morris water maze in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc supplementation. To assess the effects of genetic HBP activation we overexpressed the key enzyme GFAT1 as well as a constitutively activated mutant form in all mouse tissues. We detected elevated UDP-GlcNAc levels in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.
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Calorie restriction (CR) is to reduce food intake without malnutrition and is sometimes adopted intentionally to reduce body weight for improving health. CR has been known to prolong the lifespan in several animal models such as nematodes, flies, and mice. While it is not feasible to implement long term CR in humans, the intake of specific food substances that produce the same effect as CR can result in significant health benefits to humans. This paper highlights the need for promoting research on such calorie restriction mimetics (CRMs). This scientific opinion suggests that the promotion of research on CRMs derived from foods and food resources has the potential to enhance health and prolong life span. Collaboration between researchers from the fields of food science and those working on CRMs has the potential to promote good health and enhance human longevity in the aging society.
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Background The aim of this study was to investigate the ability of a glucosamine-containing supplement to improve locomotor functions in subjects with knee pain. Methods A randomized, double-blind, placebo-controlled, parallel-group comparative study was conducted for 16 weeks in 100 Japanese subjects (age, 51.8±0.8 years) with knee pain. Subjects were randomly assigned to one of the two supplements containing 1) 1,200 mg of glucosamine hydrochloride, 60 mg of chondroitin sulfate, 45 mg of type II collagen peptides, 90 mg of quercetin glycosides, 10 mg of imidazole peptides, and 5 μg of vitamin D per day (GCQID group, n=50) or 2) a placebo (placebo group, n=50). Japanese Knee Osteoarthritis Measure, visual analog scale score, normal walking speed, and knee-extensor strength were measured to evaluate the effects of the supplement on knee-joint functions and locomotor functions. Results In subjects eligible for efficacy assessment, there was no significant group × time interaction, and there were improvements in knee-joint functions and locomotor functions in both groups, but there was no significant difference between the groups. In subjects with mild-to-severe knee pain at baseline, knee-extensor strength at week 8 (104.6±5.0% body weight vs 92.3±5.5% body weight, P=0.030) and the change in normal walking speed at week 16 (0.11±0.03 m/s vs 0.05±0.02 m/s, P=0.038) were significantly greater in the GCQID group than in the placebo group. Further subgroup analysis based on Kellgren–Lawrence (K–L) grade showed that normal walking speed at week 16 (1.36±0.05 m/s vs 1.21±0.02 m/s, P<0.05) was significantly greater in the GCQID group than in the placebo group in subjects with K–L grade I. No adverse effect of treatment was identified in the safety assessment. Conclusion In subjects with knee pain, GCQID supplementation was effective for relieving knee pain and improving locomotor functions.
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D-Glucosamine (GlcN) is a freely available and commonly used dietary supplement potentially promoting cartilage health in humans, which also acts as an inhibitor of glycolysis. Here we show that GlcN, independent of the hexosamine pathway, extends Caenorhabditis elegans life span by impairing glucose metabolism that activates AMP-activated protein kinase (AMPK/AAK-2) and increases mitochondrial biogenesis. Consistent with the concept of mitohormesis, GlcN promotes increased formation of mitochondrial reactive oxygen species (ROS) culminating in increased expression of the nematodal amino acid-transporter 1 (aat-1) gene. Ameliorating mitochondrial ROS formation or impairment of aat-1-expression abolishes GlcN-mediated life span extension in an NRF2/SKN-1-dependent fashion. Unlike other calorie restriction mimetics, such as 2-deoxyglucose, GlcN extends life span of ageing C57BL/6 mice, which show an induction of mitochondrial biogenesis, lowered blood glucose levels, enhanced expression of several murine amino-acid transporters, as well as increased amino-acid catabolism. Taken together, we provide evidence that GlcN extends life span in evolutionary distinct species by mimicking a low-carbohydrate diet.
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Dietary restriction (DR) is an effective intervention known to increase lifespan in a wide variety of organisms. DR also delays the onset of aging-associated diseases. DR mimetics, compounds that can mimic the effects of DR, have been intensively explored. d-Allulose (d-Alu), the C3-epimer of d-fructose, is a rare sugar that has various health benefits, including anti-hyperglycemia and anti-obesity effects. Here, we report that d-Alu increased the lifespan of Caenorhabditis elegans both under monoxenic and axenic culture conditions. d-Alu did not further extend the lifespan of the long-lived DR model eat-2 mutant, strongly indicating that the effect is related to DR. However, d-Alu did not reduce the food intake of wild-type C. elegans. To explore the mechanisms of the d-Alu longevity effect, we examined the lifespan of d-Alu-treated mutants deficient for nutrient sensing pathway-related genes daf-16, sir-2.1, aak-2, and skn-1. As a result, d-Alu increased the lifespan of the daf-16, sir-2.1, and skn-1 mutants, but not the aak-2 mutant, indicating that the lifespan extension was dependent on the energy sensor, AMP-activated protein kinase (AMPK). d-Alu also enhanced the mRNA expression and enzyme activities of superoxide dismutase (SOD) and catalase. From these findings, we conclude that d-Alu extends lifespan by increasing oxidative stress resistance through a DR mechanism, making it a candidate DR mimetic.
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Caloric restriction, be it constant or intermittent, is reputed to have health-promoting and lifespan-extending effects. Caloric restriction mimetics (CRMs) are compounds that mimic the biochemical and functional effects of caloric restriction. In this Opinion article, we propose a unifying definition of CRMs as compounds that stimulate autophagy by favouring the deacetylation of cellular proteins. This deacetylation process can be achieved by three classes of compounds that deplete acetyl coenzyme A (AcCoA; the sole donor of acetyl groups), that inhibit acetyl transferases (a group of enzymes that acetylate lysine residues in an array of proteins) or that stimulate the activity of deacetylases and hence reverse the action of acetyl transferases. A unifying definition of CRMs will be important for the continued development of this class of therapeutic agents.
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Aging entails a progressive decline in protein homeostasis, which often leads to age-related diseases. The endoplasmic reticulum (ER) is the site of protein synthesis and maturation for secreted and membrane proteins. Correct folding of ER proteins requires covalent attachment of N-linked glycan oligosaccharides. Here, we report that increased synthesis of N-glycan precursors in the hexosamine pathway improves ER protein homeostasis and extends lifespan in C. elegans. Addition of the N-glycan precursor N-acetylglucosamine to the growth medium slows aging in wild-type animals and alleviates pathology of distinct neurotoxic disease models. Our data suggest that reduced aggregation of metastable proteins and lifespan extension depend on enhanced ER-associated protein degradation, proteasomal activity, and autophagy. Evidently, hexosamine pathway activation or N-acetylglucosamine supplementation induces distinct protein quality control mechanisms, which may allow therapeutic intervention against age-related and proteotoxic diseases.