Synthesis of a new [6]-gingerol analogue and its protective effect with respect to the development of metabolic syndrome in mice fed a high-fat diet

Article abstract of DOI:10.1021/jm200662c

To determine the effects of a [6]-gingerol analogue (6G), a major chemical component of the ginger rhizome, and its stable analogue after digestion in simulated gastric fluid, aza-[6]-gingerol (A6G), on diet-induced body fat accumulation, we synthesized 6

Full text of DOI:10.1021/jm200662c

ARTICLE
pubs.acs.org/jmc
Synthesis of a New [6]-Gingerol Analogue and Its Protective Effect
with Respect to the Development of Metabolic Syndrome in Mice
Fed a High-Fat Diet
||
Mayumi Okamoto,*,†, Hiroyuki Irii,† Yu Tahara,‡ Hiroyuki Ishii,§ Akiko Hirao,‡ Haruhide Udagawa,||
Masaki Hiramoto,|| Kazuki Yasuda,|| Atsuo Takanishi,§ Shigenobu Shibata,‡ and Isao Shimizu†
†Department of Applied Chemistry, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan
‡Department of Physiology and Pharmacology, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku,
Tokyo, Japan
§Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan
Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine,
Shinjuku-ku, Tokyo, Japan
ABSTRACT:
To determine the effects of a [6]-gingerol analogue (6G), a major chemical component of the ginger rhizome, and its stable analogue
after digestion in simulated gastric fluid, aza-[6]-gingerol (A6G), on diet-induced body fat accumulation, we synthesized 6G and
A6G. Mice were fed either a control regular rodent chow, a high-fat diet (HFD), or a HFD supplemented with 6G and A6G.
Magnetic resonance imaging adiposity parameters of the 6G- and A6G-treated mice were compared with those of control mice.
Supplementation with 6G and A6G significantly reduced body weight gain, fat accumulation, and circulating levels of insulin and
leptin. The mRNA levels of sterol regulatory element-binding protein 1c (SREBP-1c) and acetyl-CoA carboxylase 1 in the liver were
significantly lower in mice fed A6G than in HFD control mice. Our findings indicate that A6G, rather than 6G, enhances energy
metabolism and reduces the extent of lipogenesis by downregulating SREBP-1c and its related molecules, which leads to the
suppression of body fat accumulation.
’ INTRODUCTION
Roscoe) is widely used around the world in food as a spice. In
addition, it has been an important ingredient in Chinese,
Ayurvedic, and Tibb-Unani herbal medicines for the treatment
of catarrh, rheumatism, nervous diseases, gingivitis, toothache,
asthma, stroke, constipation, and diabetes.7ꢀ9 The major
chemical constituents of ginger rhizome are essential volatile
oils and nonvolatile pungent compounds.10,11 The volatile oil
components are mainly various terpenoids. The nonvolatile
compounds include gingerol [5-hydroxy-1-(4-hydroxy-3-
methoxyphenyl)-3-decanone], shogaol [1-(4-hydroxy-3-meth-
oxyphenyl)dec-4-en-3-one], paradol, and zingerone. Among
them, gingerol and shogaol have been identified as the primary
ginger-derived bioactive constituents.12ꢀ14 Recently, a report
demonstrated the hypoglycemic potential of ginger in strepto-
zotocin (STZ)-induced diabetic rats treated with an aqueous
extract of raw ginger daily for a period of 7 weeks; additionally,
it showed that raw ginger was effective in reversing diabetic
protein urination and loss of body weight observed in the
diabetic rats.15
Metabolic syndrome comprises a constellation of metabolic
abnormalities, including dyslipidemia (low HDL cholesterol,
high LDL cholesterol, and high triglycerides), hyperglycemia,
and elevated blood pressure, that increase the risk of developing
cardiovascular disease. Two major underlying risk factors for
metabolic syndrome are obesity and insulin resistance. Obesity
results from an imbalance between fat synthesis (lipogenesis)
and fat breakdown (lipolysis), both of which are regulated by the
level and activity of molecules involved in carbohydrate and
lipid metabolism. Thus, the molecules involved in these path-
ways are considered potential targets for the treatment of
obesity and metabolic syndrome.1,2 The expression of many
lipogenic genes, such as acetyl-CoA carboxylase (ACC) and
fatty acid synthase (FAS), is transcriptionally regulated by
sterol regulatory element-binding protein 1 (SREBP-1). Sup-
pression of lipogenic enzymes, including ACC and FAS,
attenuates body fat accumulation.1ꢀ4
The prevalence of obesity is increasing globally; therefore,
studies1,2,5,6 of drugs and functional foods that regulate energy
metabolism have been vigorously conducted with the aim of
managing obesity and related diseases. Ginger (Zingiber officinale
Received: May 25, 2011
Published: August 18, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of 6G (1) (a) and A6G (2) (b)
Gingerol and shogaol are known to activate transient receptor
potential vanilloid-1 (TRPV1).16 Therefore, gingerol and sho-
gaol may be potentially valuable in inducing weight loss. The
prototypical agonist of TRPV1 is capsaicin (8-methyl-N-vanillyl-
trans-6-nonenamide), the pungent compound found in chili
peppers. In fact, capsaicin is found to exert multiple pharmaco-
logical and physiological effects, including analgesic, anticancer,
anti-inflammatory, antioxidant, and anti-obesity effects.17 The
important structural difference between capsaicin and gingerol or
shogaol is the presence of an amide group in the former. It has
been suggested that the amide group is responsible for the anti-
obesity effects of capsaicin. Therefore, we synthesized A6G as a
representative compound of gingerol containing an amide group.
1H magnetic resonance imaging (MRI) offers several methods
for obtaining separate fat and water images and allows quantifica-
tion of fat volumes in anatomically distinct fat deposits noninva-
sively and simultaneously.18ꢀ22 The MRI system is not invasive in
itself, and this is an advantage over computed tomography (CT)
where the experimental animal must be exposed to radiation.
In this study, we synthesized a chemical analogue of the
gingerol (A6G) and examined it by comparing it with gingerol
as a potential anti-obesity therapeutic agent. We also evaluated
the effects of these compounds on energy metabolism-related
molecules and found that A6G with an amide group suppressed
body fat accumulation induced by a high-fat diet by down-
regulating SREBP-1c and downstream molecules and stimulating
the expenditure of energy in mice. Here, we have demonstrated
that the daily intake of A6G rather than 6G could be a potentially
effective and safe approach for preventing or attenuating obesity
and metabolic syndrome.
’ RESULTS
Chemical Synthesis. Structural attributes of each analogue
are presented in Scheme 1.
Effect of A6G on Fasting Blood Glucose. Blood glucose
levels after mice had fasted for 12 h were measured 0, 9, 16, 23,
30, 44, 48, 57, 68, and 86 days after mice had been fed 6G or A6G.
As shown in Figure 1a, the fasting blood glucose level prior to the
treatments among the experimental groups varied within the
normal physiological range, although the levels were significantly
different among some of the groups. At the end of 86 days, mice
fed HFD alone showed significant (P < 0.001) elevations in
fasting blood glucose levels as compared with the pretreatment
glucose levels or with those in the normal controls fed RC.
Treatment of 6G or A6G with HFD produced a significant
reduction (P < 0.001) in fasting blood glucose levels as compared
with the controls fed a HFD. Moreover, at the end of the
treatment, the serum glucose levels in the 6G- or A6G-treated
groups were not significantly different from their pretreatment
glucose levels or from those in the RC control group.
Effect of A6G on Body Weight. The changes in the mean
body weight of the experimental groups of mice over the 12-week
treatment period are shown in Figure 2b. There was no sig-
nificant difference in the initial body weights and the
body weights after 3 weeks [from 29.0 ( 0.5 to 30.3 ( 0.6 g
(n = 6)] in the different groups. Animals treated with HFD alone
exhibited significant increases in body weight (51.2 ( 2.7 g)
as compared with their pretreatment values (29.8 ( 1.6 g)
(P < 0.001) or with the control group receiving RC diet alone
(45.0 ( 0.8 g) (P < 0.001). The HFD groups treated with 6G or
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Figure 1. Structures of [6]-gingerol (6G) (1), aza-[6]-gingerol (A6G) (2), and [6]-shogaol (6S).
A6G showed significant reductions in body weight [from 51.2 (
2.7 to 47.1 ( 1.1 g (P < 0.001) for 6G and from 51.2 ( 2.7 to
41.6 ( 0.2 g (P < 0.001) for A6G] compared with HFD-treated
controls. Therefore, the body weights of the A6G-treated mice
(41.6 ( 0.2 g) were not significantly different from those of the
RC control mice after 86 days (45.0 ( 0.8 g) (P = 0.07).
Effect of A6G on VS and SC Fat Volumes. After treatment
with each compound for 86 days, MRI was performed. Figure 3
shows a 1H fat MRI image of representative different groups.
Groups treated with HFD alone exhibited a significant
increase (20.8 ( 1.7%) in percent body fat [visceral (VS)
and subcutaneous (SC)] as compared with the RC control
group (5.4 ( 1.7%) (P < 0.001). The HFD groups treated
with 6G or A6G showed significant reductions in percent
body fat [from 20.8 ( 1.7 to 10.9 ( 1.0% (P < 0.001) for A6G
and from 20.8 ( 1.7 to 6.7 ( 0.8% (P < 0.001 for 6G] as
compared with the HFD-treated controls, whereas the per-
cent body fat of A6G-treated mice (6.7 ( 0.8%) was not
significantly different from the RC control mice after 86 days
(5.4 ( 1.7%) (P = 0.41).
Figure 4b shows the changes in the VS fat volumes of different
groups under sacrificed conditions. The HFD animals treated
with 6G or A6G showed a significant decrease [3.5 ( 0.2 g
(P < 0.01) for 6G and 1.84 ( 0.2 g (P < 0.001) for A6G] in the
VS fat volumes as compared with controls treated with HFD
alone (5.03 ( 0.5 g). A significant difference was noted
between the 6G group (3.5 ( 0.2 g) and the A6G group for
VS fat weight (1.84 ( 0.2 g) (P < 0.001). No statistically
Figure 2. Effect of 6G or A6G treatment for 86 days on (a) fasting blood
glucose level and (b) body weight in HFD-induced mice. Data are
expressed as means ( standard error of the mean (n = 5 or 6; ***P <
0.001 vs HFD).
significant difference was found between the adiposity volumes
calculated via MRI and the adiposity volumes found at autopsy
(for all, P > 0.2).
Effect of A6G on the Oral Glucose Tolerance Test. On days
92ꢀ94, OGTT was performed. Treatment with 6G or A6G
significantly (P < 0.001) improved the glucose tolerance and
inhibited the increase in postprandial glucose levels after glucose
load intake in the groups fed HFD alone (Figure 4c).
Effect of A6G on Serum Hormones and Gene Expression
in Adipose and Liver Tissue. Prior to the treatments with the
compounds, the serum insulin concentration was not signifi-
cantly different among the experimental groups. However, the
groups treated with 6G or A6G showed a significant reduction in
serum insulin levels (P < 0.05 for 6G; P < 0.01 for A6G)
(Figure 5a) and serum leptin levels (P < 0.01) (Figure 5b) as
compared with HFD-treated controls. Serum insulin and leptin
concentrations were lower, but not significantly, in the A6G-
treated groups than in the 6G-treated groups. Supplementation
with 6G or A6G significantly reduced the level of mRNA
expression of leptin (Figure 5c) and TNFα (Figure 5d) in
adipose tissue (P < 0.01). The level of mRNA expression of
leptin was lower, but not significantly, in the A6G groups as
compared to that in the 6G groups.
To elucidate the underlying mechanisms of the effects of A6G,
we used real-time polymerase chain reaction (PCR) after mice
had been fed for 86 days, during which time there were no
significant differences in food intake among the HFD groups, to
examine A6G-induced changes in gene expression in the liver.
Supplementation with 6G or A6G caused insignificant decreases
in the mRNA levels of ACC-1 and FAS, which are lipogenic
enzymes in the liver, as compared with the respective levels in the
HFD control group (Figure 6b,c). The mRNA levels of SREBP-
1c, the master regulator of fatty acid synthesis,23,24 were lower in
the HFD group treated with A6G (Figure 6a).
A6G Attenuates TNFα-Mediated Downregulation of adi-
ponectin Gene Expression. Marked reduction of the level of
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Figure 3. Axial images of mice fed RC (a and b), HFD (c and d), HFD with 6G (e and f), or HFD with A6G (g and h). Panels a, c, e, and g show
subcutaneous fat (SC), and panels b, d, f, and h show visceral fat (VS).
adiponectin gene expression was observed after the administra-
tion of TNFα (P < 0.0001) (Figure 7) in adipocytes. In contrast,
downregulation of adiponectin gene expression was significantly
attenuated by pretreatment with 6G or A6G (P < 0.01 for 25 and
50 μM 6G, P < 0.05 for 25 μM A6G, and P < 0.001 for 50 μM
A6G) (Figure 7) as well as with rosiglitazone (P < 0.0001).
Furthermore, the adiponectin gene expression after pretreatment
with 50 μM A6G (0.793 ( 0.14) was not significantly different
from that after pretreatment with rosiglitazone (0.879 ( 0.21)
(P = 0.939; Figure 7).
prophylactic effects of chronic treatment with 6G and/or A6G.
To the best of our knowledge, this is the first study to use mice
fed a high-fat diet to investigate the protective effects of gingerol
derivatives in vivo.
Metabolic syndrome is a complex polygenic disorder resulting
in part from the contribution of impaired insulin secretion and/
or impaired insulin action on its receptors.26 When carbohy-
drates are in short supply, or their breakdown is incomplete, fats
become the preferred source of energy.27 As a result, fatty acids
are mobilized into the general circulation, leading to secondary
triglyceridemia with increased levels of total serum lipids, in-
cluding triglycerides, cholesterol, and phospholipids, leading to
life-threatening lipid disorders.28 The development of metabolic
syndrome is influenced by a combination of genetic and envir-
onmental factors. Among the environmental factors, long-term
high-fat intake has been most intensively studied because of its
contribution to the development of metabolic syndrome in hu-
mans and rodents.29 Apparently, a high proportion of daily energy
’ DISCUSSION
In this study, we examine the protective effects of 6G and A6G
in mice fed a high-fat diet, a metabolic model of obesity and type
2 diabetes that is similar to human metabolic syndrome.25
Because mice have metabolic patterns similar to those of
humans, it is rational to use this disease model to examine the
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Figure 4. Effect of treatment with 6G or A6G for 86 days on (a) body weight, (b) epididymal fat weight, and (c) oral glucose tolerance. Data are
expressed as means ( standard error of the mean (n = 5 or 6; **P < 0.01 or ***P < 0.001 vs HFD).
derived from the fat component is a common situation in current
lifestyles in most societies of the world. The high prevalence of
metabolic disorders is probably related to abnormal blood profiles
that may be due to the long-term effects of high-fat intake.30
Significant increases in body weight, serum glucose levels, and
insulin levels along with reduced insulin sensitivity have been
demonstrated in mice fed a high-fat diet.14,31,32 This study reveals
that 6G and A6G effectively suppressed body weight gain and
body fat accumulation, lowered serum glucose and insulin levels,
and increased insulin sensitivity upon concomitant administra-
tion along with high-fat diet.
The data obtained in this study are in agreement with a recent
report that 6G significantly inhibited the TNFα-mediated down-
regulation of adiponectin gene expression in adipocytes.13 In
addition, we demonstrated that A6G significantly inhibited that
process like 6G. Isa et al.13 reported an upregulation of adipo-
nectin by [6]-shogaol (6S) and 6G, the major components of Z.
officinale. They also reported that 6S, but not 6G, possesses
significant PPAR-γ agonistic activity. It is clear that the plasma
adiponectin concentration and its mRNA expression level both
undergo a reduction in obese and insulin-resistant states; it is
therefore believed that the exogenous administration or upregula-
tion of endogenous adiponectin by pharmacological intervention
would improve insulin sensitivity, followed by an increase in the
level of fatty acid oxidation and a decrease in serum triglyceride
levels.13 Thus, these latter findings strongly lend support to the
results of our study involving mice treated with a high-fat diet,
suggesting that the glucose-regulating and insulin-sensitizing
effects of 6G and A6G can, at least in part, be attributed to
PPAR-γ agonist activity and/or the upregulation of adiponectin.
We observed that 6G and A6G downregulated lipogenic
enzymes, such as ACC-1 and FAS, in the liver. The expression
of several lipogenic genes is regulated by SREBP-1c at the
transcriptional level.23,24 According to our findings, intake of
A6G decreased SREBP-1c mRNA levels in the liver. Overall, our
results suggest that A6G represses the lipogenic pathway by
downregulating SREBP-1c, which leads to a reduction in the rate
of accumulation of body fat.
Gingerols possess a labile hydroxyketo functional group, which
renders them susceptible to transformation to less pungent
compounds such as shogaols and zingerone at elevated
temperatures.12,33 The stability of 6G and 6S is significant and
relevant as these substances are generally considered the main
active constituents in ginger-based medicinal products. In the
gastrointestinal tract, gingerols are exposed, in addition to acid, to
digestive juices, containing pepsin in the stomach and pancreatin
in the small intestine. Exposure of gingerols to these compounds
could lead to considerable acid- and/or enzyme-catalyzed
decomposition.34 It is known that biotransformation of drugs in
biological systems consists of reactions such as oxidation, reduc-
tion, hydrolysis, and conjugation of a drug or its metabolites,
leading to activation or inactivation of the drug or formation of
metabolites with pharmacological activities different from those
of the parent drug. Isa et al.13 reported that the two ginger-derived
components have a potent and unique pharmacological function
in 3T3-L1 adipocytes via different mechanisms. A recent study
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Figure 5. Effect of 6G or A6G on HFD-induced metabolic changes, (a) serum insulin and (b) leptin, and mRNA expression in adipose tissue of
(c) leptin and (d) TNFα. Data are expressed as means ( standard error of the mean (n = 3ꢀ6; *P < 0.05, **P < 0.01, and ***P < 0.001 vs HFD).
demonstrated that ginger-derived components do havesignificant
potency and act in a unique pharmacological manner that is
responsible for the regulation of adipocyte function. Both 6S and
6G significantly inhibited TNFα-mediated downregulation of
adiponectin expression in adipocytes. 6S, but not 6G, is a
significant agonist for PPAR-γ. The significant upregulation of
adiponectin observed after administration of 6S supports the
finding that 6S inhibits TNFα-mediated downregulation of
adiponectin expression by functioning as a PPAR-γ agonist.
Ginger-derived components (6S and 6G) have a unique ther-
apeutic potential that possibly may be used to regulate adipocyte
function. These compounds can significantly inhibit TNFα-
mediated downregulation of adiponectin in adipocytes, although
they do so via different mechanisms.
We found that A6G was stable in simulated gastric fluid
(pH 1) or 6 N HCl from 25 to 140 °C without degradation
leading to the formation of shogaols. However, in simulated
gastric fluid, 6G and 6S underwent first-order reversible dehy-
dration and hydration reactions to form 6S and 6G, respectively.
The presence of the hydroxyl moiety (6G) does not affect PPAR-
γ activation, although it does contribute to the inhibition of
TNFα-mediated JNK activation. In the absence of the hydroxyl
moiety (6S), the compound is effective as a functional PPAR-γ
agonist; however, there is diminished inhibitory activity of JNK
activation.13 In short, the structure, 6S or 6G, that possesses anti-
obesity activity when administered orally remains unclear. Be-
cause A6G is a stable structure, we hope to elucidate the
mechanisms of its anti-obesity effects in our future studies. In
conclusion, the new [6]-gingerol analogue, A6G, which includes
an amide group, revealed a remarkable protective effect against
high-fat diet-induced metabolic disturbances by strongly
suppressing body weight gain, hyperglycemia, and body fat
accumulation by modulating fatty acid or glucose metabolism
and insulin resistance conditions. Moreover, A6G remained
stable in simulated gastric fluids (pH 1) at 37 °C. Thus, our
findings suggest that A6G possesses potential therapeutic value
and could potentially reduce the risk of obesity-associated
disease, including type 2 diabetes. Further studies are being
undertaken for achieving an in-depth understanding of the
mechanism(s) underlying the glucose and lipid metabolism-
regulating activities of A6G.
’ MATERIALS AND METHODS
All reagents and solvents were purchased commercially and used as
received. 1H NMR and 13C NMR spectra were recorded with CDCl3 as a
solvent using tetramethylsilane as an internal standard on JEOL AL-400
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starting with ethyl 3-(tert-butyldimethylsilyloxy)octanoate 336 as shown
in Scheme 1b.
5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone
(1). To a stirred solution of Zingerone (3, 1941 mg, 10 mmol) and t-
BuOK (2805 mg, 25 mmol) in THF (100 mL) was added hexanal
(1.48 mL, 12 mmol) at ꢀ78 °C. After 1.5 h, the reaction was quenched
by the addition of saturated aqueous NH4Cl. The mixture was extracted
with ethyl acetate. The combined organic layer was washed with brine,
dried over MgSO4, filtered, and evaporated to dryness under reduced
pressure. The crude product was purified by flash column chromatog-
raphy (12.5% ethyl acetate/hexane) to provide 1 (2246 mg, 76%): 1H
NMR δ 0.88 (s, 3H), 1.21ꢀ1.53 (m, 8H), 2.48 (dd, 1H, J = 17.4 Hz, J =
8.9 Hz), 2.57 (dd, 1H, J = 17.4 Hz, J = 3.2 Hz), 2.77 (m, 4H), 2.93 (s,
1H), 3.88 (s, 3H), 4.02 (m, 1H), 5.56 (s, 1H), 6.67 (m, 2H), 6.83 (d, 1H,
J = 8.0 Hz); 13C NMR δ 14.0, 22.6, 25.1, 29.3, 31.7, 36.4, 45.4, 49.3, 55.9,
111.0, 114.3, 120.7, 132.0, 144.0, 146.4, 211.4; IR (neat) 3423.6 (br),
2931.7 (s), 2858.5 (m), 1704.6 (s), 1516.4 (s), 1272.1 (m), 1236.9 (m),
1154.1 (m), 1034.7 (m) cmꢀ1; HRMS (ESI) m/z calcd for C16H27O4
(M) 294.1831, found 294.1825.
Ethyl 3-(tert-Butyldimethylsilyloxy)octanoate (5). To a stir-
red solution of dimethylaminopyridine (DMAP, 39.2 mg, 0.32 mmol),
imidazole (0.871 g, 12.7 mmol), and tert-butyldimethylchlorosilane
(1.45 g, 9.6 mmol) was added alcohol 4 (0.602 g, 3.2 mmol) in DMF
(32 mL), and the mixture was stirred for 6 h. Saturated aqueous NH4Cl
was added to the reaction mixture, and the mixture was extracted with
ether. The ethereal extract was washed with brine and a phosphate buffer
solution, dried over anhydrous magnesium sulfate, filtered, and con-
densed in vacuo. The residue was chromatographed on silica gel with a
3% ethyl acetate/hexane solution as an eluent to provide silyl ether 5
(0.851 g, 88%): 1H NMR δ 0.035 (s, 3H), 0.059 (s, 3H) 0.87 (s, 9H),
0.89 (t, 3H, J = 6.8 Hz), 1.27 (t, 3H, J = 7.1 Hz), 1.3ꢀ1.5 (m, 8H), 2.40
(dd, 1H, J = 14.6 Hz, J = 5.9 Hz), 2.42 (dd, 1H, J = 14.6 Hz, J = 7.1 Hz),
4.11 (m, 1H), 4.12 (q, 2H, J = 7.1 Hz); 13C NMR δ ꢀ4.75, ꢀ4.47, 14.0,
14.2, 18.1, 22.7, 24.7, 25.9, 31.9, 37.6, 42.8, 60.3, 69.5, 172.0; IR (neat)
2956.8 (m), 2930.5 (s), 2857.9 (m), 1739.1 (s), 1255.4 (w), 1094.0 (w),
836.3 (m), 775.6 (w) cmꢀ1; HRMS (ESI) m/z calcd for C16H34O3SiNa
(M + Na)+ 325.2175, found 325.217.
3-(tert-Butyldimethylsilyloxy)octanoic Acid (6). To a solution
of sodium hydroxide (0.5 g, 12.5 mmol) in water (8 mL) was added ester
5 (0.756 g, 2.5 mmol) in methanol (2 mL), and the mixture was stirred
for 24 h at room temperature. Dilute hydrochloric acid (1 M) was added
to the mixture, and the mixture was extracted with ether. The combi-
ned organic layer was dried over anhydrous magnesium sulfate, filtered,
and condensed in vacuo. The residue was chromatographed on silica gel
with a solution of 15% ethyl acetate in hexane as an eluent to provide 6
(0.584 g, 85%): 1H NMR δ 0.104 (s, 3H), 0.113 (s, 3H), 0.89 (t, 3H, J =
5.8 Hz), 0.90 (s, 9H), 1.3ꢀ1.5 (m, 8H), 2.49 (dd, 1H, J = 15.1 Hz, J = 5.3
Hz), 2.57 (dd, 1H, J = 15.1 Hz, J = 5.1 Hz), 4.11 (quintet, 1H, J = 6.0 Hz);
13C NMR δ ꢀ4.88, ꢀ4.56, 14.0, 18.0, 22.6, 24.7, 25.7, 31.8, 37.3, 42.2,
69.4, 177.5; IR (neat) 2956.9 (m), 2930.7 (m), 2858.7 (m), 1713.3 (s),
1255.4 (w), 1094.0 (w), 836.3 (m), 775.6 (w) cmꢀ1; HRMS (ESI) m/z
calcd for C14H30O3SiNa (M + Na)+ 297.1862, found 297.1869.
3-(tert-Butyldimethylsilyloxy)-N-(4-hydroxy-3-methoxy-
benzyl)octanamide (8). To a stirred solution of carboxylic acid 6
(0.150 g, 0.55 mmol) in dichloromethane (2.5 mL) were added N,N-
dimethyl-4-aminopyridine (6.84 mg, 0.056 mmol), N,N-dicyclohexyl-
carbodiimide (0.227 g, 1.1 mmol), N-hydroxysuccinimide (0.127 g,
1.1 mmol), N,N-diisopropylethylamine (0.29 mL, 1.65 mmol), and
ammonium chloride 7 (0.126 g, 0.67 mmol) in N,N-dimethyl sulfoxide
(2.5 mL), and the mixture was stirred for 48 h. A saturated aqueous
NH4Cl solution was added to the mixture, and the mixture was
extracted with ether. The ethereal extract was dried over anhydrous
magnesium sulfate, filtered, and condensed in vacuo. The residue was
chromatographed on silica gel with a solution of 15% ethyl acetate in
Figure 6. Effect of 6G or A6G on HFD-induced mRNA expression in
liver of (a) SREBP-1c, (b) ACC, and (c) FAS. Data are expressed as
means ( standard error of the mean (n = 3ꢀ6; *P < 0.05 vs HFD).
spectrometers. Multiplicities are indicated as br (broadened), s (singlet),
d (doublet), t (triplet), q (quartet), or m (multiplet). Infared (IR)
spectra were recorded on a JEOL JIR-WINSPECK 50 FT-IR spectro-
meter (νmax in cmꢀ1). Bands are characterized as br (broad), s (strong),
m (medium), or w (weak). High-resolution mass spectrometry
(HRMS) spectra were recorded on a JMS-SX102A instrument (JEOL,
Tokyo, Japan). Gas chromatographic analyses (GC) were conducted on
a GC-4000 instrument (GL Sciences, Tokyo, Japan) equipped with an
InsertCap 1 column [0.53 mm (inside diameter) ꢁ 30 m], using He as a
carrier gas. Purity was determined by NMR and GC, and all compounds
were confirmed to be >98% pure. [6]-Gingerol (6G) (1) was prepared
as a racemic mixture according to a previously described method35
(Scheme 1a). Synthesis of aza-[6]-gingerol (A6G) (2) was conducted by
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Figure 7. Inhibitory effect of 6G or A6G on the TNFα-mediated downregulation of adiponectin gene expression in 3T3-L1 adipocytes. Mice fed 6G
(10, 25, and 50 μM), A6G (10, 25, and 50 μM), rosiglitazone (10 μM), or control (0.5% dimethyl sulfoxide) were treated for 1 h before being treated
with TNFα at a concentration of 10 ng/mL for 15 h. Data are expressed as means ( standard error of the mean (n = 8). Values without a common letter
are significantly different at the P < 0.05 level.
1
hexane as an eluent to provide amide 8 (94.5 mg, 42%): H NMR δ
0.035 (s, 3H), 0.047 (s, 3H), 0.80 (s, 9H), 0.88 (t, 3H, J = 6.9 Hz),
1.3ꢀ1.5 (8H), 2.31 (dd, 1H, J = 14.7 Hz, J = 5.1 Hz), 2.47 (dd, 1H, J =
14.7 Hz, J = 4.2 Hz), 3.87 (s, 3H), 4.01 (m, 1H), 4.23 (dd, 1H, J = 14.3
Hz, J = 5.1 Hz), 4.50 (dd, 1H, J = 14.3 Hz, J = 6.0 Hz), 6.77 (dd, 1H, J =
8.06 Hz, J = 1.83 Hz), 6.80 (d, 1H, J = 1.83 Hz), 6.85 (d, 1H, J = 8.06
Hz); 13C NMR δ ꢀ4.90, ꢀ4.76, 13.9, 17.7, 22.5, 25.1, 25.6, 31.7, 36.6,
43.4, 55.8, 69.7, 110.9, 114.5, 120.9, 130.0, 145.1, 146.7, 174.0; IR (neat)
3306.7 (br), 2955.6 (m), 2930.0 (s), 2857.4 (m), 1651.7 (s), 1516.7 (s),
1463.5 (m), 1432.9 (w), 1378.3 (w), 1255.9 (m), 836.5 (w), 776.5
(w) cmꢀ1; HRMS (ESI) m/z calcd for C22H39NO4SiNa (M + Na)+
432.2546, found 432.2564.
ad libitum, or as otherwise noted. The light intensity at the surface of the
cages was approximately 100 lx. Mice were fed either a control regular
rodent chow, i.e., 63% carbohydrate, 5% fat, and 32% protein (CE-2,
CLEA Japan), or a high-fat diet, i.e., 25% beef tallow (w/w) added to the
regular rodent chow (Oriental Yeast Co., Ltd.), or a high-fat diet
supplemented with 0.06% 6G or A6G for 86 days. The animal experi-
ments were conducted under the permission of the Animal Welfare
Committee of Waseda University (Permission 10A35).
Experimental Protocol. The mice were randomly divided into
groups of 6 each on day 0. The fasting blood glucose level was measured
after animals had fasted for 12 h (starting from 8:00 p.m.) on day 0
(before treatment), 9, 16, 23, 30, 44, 48, 57, 68, and 86. On day 90, MRI
was performed. On days 92ꢀ94, an oral glucose tolerance test (OGTT)
was performed after overnight fasting. Blood was sampled from the tail
vein of mice at 0 min (baseline) and 30, 60, 120, and 240 min after an oral
glucose load of 3.0 g/kg of body weight. Food, but not water, was
withheld from the cages during the OGTT test. Blood glucose levels were
measured by Glucose Pilot (Aventir Biotech, LLC) as per the manufac-
turer’s instructions. On day 100, after having fasted for 12 h, the mice
from the four groups (HFD, RC, HFD with A6G, and HFD with 6G)
were sacrificed; the visceral and subcutaneous fat and liver were dissected,
and blood samples were collected. Plasma samples were stored at ꢀ20 °C
for the determination of insulin and leptin levels using the mouse insulin
ELISA kit (Shibayagi Co., Ltd., Gunma, Japan) and the mouse leptin
ELISA kit (R&D, Minneapolis, MN), respectively, according to the
manufacturers’ instructions. The visceral fat obtained from each mouse
was dissected and weighed. A small portion of the liver and the visceral fat
from each mouse was dissolved in phenol (Omega Biotek, Inc.).
RNA Isolation and Real-Time RT-PCR. Total RNA was ex-
tracted using the phenol-dissolved samples. Here, 50 ng of total RNA
was reverse transcribed and amplified using the One-Step SYBR RT-
PCR Kit (TaKaRa, Otsu, Japan) in the Step One Plus Real-Time PCR
System (Applied Biosystems, Foster City, CA). Specific primer pairs
were designed using Primer3 Input (version 0.4.0). The following
primers were used: β-actin-F, 50-TGACAGGATGCAGAAGGAGA-30;
3-Hydroxy-N-(4-hydroxy-3-methoxybenzyl)octanamide
(2). To a solution of 7 (92.1 mg, 0.22 mmol) in CH2Cl2 (2.2 mL) was
added p-toluenesulfonic acid (0.129 g, 0.68 mmol), and the mixture was
stirred for 2 h. A saturated aqueous NaHCO3 solution was added to the
mixture, and the mixture was extracted with dichloromethane. The
extract was dried over anhydrous magnesium sulfate, filtered, and
condensed in vacuo. The residue was chromatographed on silica gel
with a 2:1 ethyl acetate/hexane solution as an eluent to provide 2 (47.0
mg, 72%): 1H NMR δ 0.88 (t, 3H, J = 6.6 Hz), 1.3ꢀ1.5 (8H), 2.27 (dd,
1H, J = 15.2 Hz, J = 8.8 Hz), 2.38 (dd, 1H, J = 15.2 Hz, J = 2.9 Hz), 4.00
(m, 1H), 4.36 (d, 1H, J = 5.5 Hz), 6.77 (dd, 1H, J = 7.7 Hz, J = 1.7 Hz),
6.80 (d, 1H, J = 1.7 Hz), 6.87 (d, 1H, J = 7.7 Hz); 13C NMR δ 23.7, 32.2,
34.8, 41.4, 46.6, 52.2, 53.1, 65.6, 78.5, 120.4, 124.2, 130.4, 139.5, 154.8,
156.5, 182.0; IR (neat) 3318.7 (br), 2930.7 (m), 2857.6 (s), 1638.2 (s),
1550.3 (w), 1517.1 (s), 1464.3 (m), 1431.8 (w), 1238.6 (w), 1277.0
(m), 1034.8 (m), 835.2 (w), 798.5 (w) cmꢀ1; MS (FAB) M = 295.2,
296.2 (M + 1); HRMS (ESI) m/z calcd for C16H25NO4Na (M + Na)+
318.1681, found 318.1692.
Animals. Male Jcl:MCH (ICR) mice [n = 21, body weight of 27ꢀ
30 g, 6 weeks of age (CLEA Japan, Tokyo, Japan)] were used in this
study. The animal room had a controlled temperature of 22 ( 2 °C, a
humidity of 60 ( 5%, and a 12 hꢀ12 h lightꢀdark cycle (i.e., lights
on from 8:00 a.m. to 8:00 p.m.); the animals were given food and water
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β-actin-R, 50-GCTGGAAGGTGGACAGTGAG-30; Gapdh-F, 50-
TGGTGAAGGTCGGTGTGAAC-30; Gapdh-R, 50-AATGAAGGGG-
TCGTTGATGG-30; TNFα-F, 50-ATCCGCGACGTGGAACTG-30;
TNFα-R, 50-ACCGCCTGGAGTTCTGGAA-30; leptin-F, 50-TCACA-
CACGCAGTCGGTATC-30; leptin-R, 50-GCTGGTGAGGACCTGT-
TGATAG-30; SREBP-1c-F, 50-CGCTACCGGTCTTCTATCAATG-
30; SREBP-1c-R, 50-CAAGAAGCGGATGTAGTCGATG-30; ACCα-
F, 50-CATGAACACCCAGAGCATTG-30; ACCα-R, 50-ATTTGTCG-
TAGTGGCCGTTC-30; FAS-F, 50-GGCAGAGAAGAAAGCTGTGG-
30; and FAS-R, 50-TCGGATGCCTAGGATGTGTG-30 [with forward
(F), reverse (R), glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
tumor necrosis factor (TNF), acetyl-CoA (ACC), and fatty acid synthase
(FAS)]. RT-PCR was performed under the following conditions: cDNA
synthesis at 42 °C for 15 min followed by 95 °C for 2 min, PCR
amplification for 40 cycles with denaturation at 95 °C for 5 s, and
annealing and extension at 60 °C for 20 s. The relative light unit of the
target gene PCR products was normalized to that of β-actin for liver
samples or Gapdh for adipose tissue samples. Melting curve analysis was
then performed. The specificity of gene amplification was confirmed by
measuring the size and purity of the PCR product by gel electrophoresis.
3T3-L1 Cell Culture and Treatment. Mouse 3T3-L1 cells were
purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). These
cells were cultured in preadipocyte maintenance medium (DS Pharma
Biomedical) at 37 °C in 5% CO2. Confluent cells were placed in a
differentiation medium (DS Pharma Biomedical) for 3 days. The
medium was then changed to adipocyte maintenance medium (DS
Phama Biomedical). Seven days after the differentiation of preadipocytes
into adipocytes, these adipocytes were pretreated with 6G, A6G,
rosiglitazone (rosiglitazone is a thiazolidinedione that has been shown
to be highly effective in reducing insulin resistance and improving type 2
diabetes) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) or
vehicles (0.5% dimethyl sulfoxide) for 1 h. Then the cells were treated
with TNFα [10 ng/mL (Sigma-Aldrich, St. Louis, MO)] for 15 h. After
treatment, the total RNA from the adipocytes was isolated using the
QIAzol TM reagent (QIAGEN, Tokyo, Japan) according to the manu-
facturer’s directions. cDNA was prepared from 1 μg of total RNA using
reverse transcriptase [Superscript III (Invitrogen)]. The quantification of
gene expression in the adipocytes was measured using TaqMan Gene
Expression Assays (mouse adiponectin, Mm00456425_m1; mouse β-
actin, 4352933E), the TaqMan fast advanced master mix core reagent kit,
and ABI Prism 7900 (Applied Biosystems). The gene expression level
was evaluated by the 2(ꢀΔΔC(T)) method.37 The gene expression level
was expressed relative to the control (1.0) after normalization using the
β-actin gene expression level.
Magnetic Resonance Imaging. An exact quantification of fat
distribution was performed by MRI as previously described by Machann
et al.38,39 MRI-derived visceral (VS) fat, subcutaneous (SC) fat, and total
body fat masses are represented in milligrams. The mice were imaged on
a 0.4 T open MRI system (Hitachi Medico Ltd., Tokyo, Japan) using a
custom-designed sendꢀreceive coil assembled to take high-resolution
images. This is a solenoid-type system, and its resonance frequency is
tuned to fit that of the MRI system using a network analyzer. Using this
coil, a clearer image can be obtained compared to that obtained using
conventional MRI receiver coils. The experimental animal was adminis-
tered an anesthetic before being placed into the MRI and then into the
coil. Axial spin-echo T1-weighted sequences were obtained for the entire
mouse (head to anus). The distribution of the fat was visualized with
Osirix version 3.9. Volumes of the VS and SC fats and the whole body of
the animal were measured using three-dimensional (3D) volume-
rendering software (Mimics 13.0, Materialize). The procedure can be
described in the following three steps: (1) areas of fat and the whole
body extracted and binarized using the binarizing function provided by
the software with a threshold for fat, (2) volumes of the fats and the
whole body calculated using the 3D volume rendering function provided
by the software, and (3) percent body fat calculated.
Statistical Analyses. The data were expressed as means (
standard error of the mean. The differences among the means were
analyzed by a Tukey-Kramer test following one-way ANOVA. Differ-
ences in P values of <0.05 were considered statistically significant. All the
statistical analyses were performed using StatView version 5.0 (SAS
Institute Inc., Cary, NC).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +81-3-5286-3205. Fax: +81-3-3232-5270. E-mail: okamoto@
aoni.waseda.jp.
’ ACKNOWLEDGMENT
The MRI receiving coil was developed with the support by Prof.
Iseki and Dr. Suzuki (Tokyo Women’s Medical University, Tokyo,
Japan). This research was supported by the Supporting Project to
Form the Strategic Research Platforms for Private University and a
Matching Fund Subsidy from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), Japan.
’ ABBREVIATIONS USED
HFD, high-fat diet;RC, regular chow; SREBP, sterol regulatory
element-binding protein;HDL, high-density lipoprotein; LDL,
low-density lipoprotein;ACC, acetyl-CoA carboxylase;FAS, fatty
acid synthase; STZ, streptozotocin;TRPV1, transient receptor
potential vanilloid-1; MRI, magnetic resonanceimaging; CT, com-
puted tomography; OGTT, oral glucose tolerance test; VS, visceral;
SC, subcutaneous; TNFα, tumor necrosis factor-α; PPAR-γ,
peroxisome proliferator-activated receptor-γ; JNK, Jun-N-
terminal kinase
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