Nutrient Interactions and Toxicity

Garlic Supplementation Increases Testicular Testosterone and Decreases

Plasma Corticosterone in Rats Fed a High Protein Diet
Yuriko Oi,1 Mika Imafuku, Chiaki Shishido, Yutaka Kominato,*
Syoji Nishimura* and Kazuo Iwai
Laboratory of Nutrition Chemistry, Faculty of Home Economics, Kobe Women’s University,
Suma-ku, Kobe 654-8585, Japan and *Riken Chemical Industry Limited Company,
Fushimi-ku, Kyoto 612-8404, Japan

ABSTRACT The effects of garlic supplementation on protein metabolism were investigated by measuring testis
testosterone and plasma corticosterone in rats fed diets with different protein levels. In Experiment 1, rats were fed
experimental diets with different protein levels (40, 25 or 10 g/100 g casein) with or without 0.8 g/100 g garlic
powder. After 28 d of feeding, testosterone contents in the testis were significantly higher and plasma corticoste-
rone concentrations were significantly lower in rats fed 40 and 25% casein diets with garlic powder than in those

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fed the same diets without garlic powder. Urinary excretion of 17-ketosteroid (an index of testosterone), nitrogen
balance and hepatic arginase activity were significantly higher in rats fed the 40% casein diet with garlic powder
than in the 40% casein controls. In Experiment 2, the effect of diallyldisulfide (a major volatile sulfur-containing
compound in garlic) on the secretion of luteinizing hormone (LH) from the pituitary gland, which regulates
testosterone production in the testis, was investigated in anesthetized rats. Plasma LH concentration increased
dose dependently after administration of diallyldisulfide (P ⬍ 0.01, r ⫽ 0.558). These results suggest that dietary
supplementation with 0.8 g/100 g garlic alters hormones associated with protein anabolism by increasing testicular
testosterone and decreasing plasma corticosterone in rats fed a high protein diet. J. Nutr. 131: 2150 –2156, 2001.

KEY WORDS: ● garlic ● diallyldisulfide ● testosterone ● corticosterone ● rats

Garlic has long been used as a spice and has been reported
to possess medicinal and pharmacologic properties. Several
studies have indicated that garlic has hypoglycemic, anticoag-
ulative, antihypertensive and hypolipidemic effects (1– 6).
However, the effects of garlic supplementation on protein
metabolism have not been fully clarified. In our previous work,
we reported that the supplementation of garlic powder at 0.8
g/100 g to a high fat diet and the administration of diallyl-
disulfide, a major volatile sulfur-containing compound in gar-
lic, enhanced triglyceride catabolism and growth of interscap-
ular brown adipose tissue (IBAT)2 by increasing noradrenaline
secretion in rats (7,8). Recently, we reported that allyl-con-
taining sulfides in garlic increase the uncoupling protein
(UCP) content in brown adipose tissue, and noradrenaline
and adrenaline secretion in rats (9). We speculated that garlic
may affect whole-body protein metabolism by the stimulation
of hormone secretion and that dietary supplementation of
garlic may enhance hormone-regulated protein anabolism.
Testosterone plays a major role in protein anabolism (10,11).
In contrast, glucocorticoid, which is secreted mainly as corti-
costerone, affects protein catabolism in rats (10). The present
study was conducted to investigate the effects of garlic supple-
1
To whom correspondence should be addressed.
E-mail: oi@suma.kobe-wu.ac.jp.
2
Abbreviations used: IBAT, interscapular brown adipose tissue; LH, luteiniz-
ing hormone; UCP, uncoupling protein.
mentation on protein metabolism in rats. In particular, we
wanted to determine whether garlic supplementation stimu-
lates protein anabolism via regulation by steroid hormones
with counterregulatory effects on both protein anabolism and
catabolism, i.e., the protein anabolic hormone, testosterone,
and the protein catabolic hormone, corticosterone. Further-
more, to better understand the effects of garlic supplementa-
tion on protein metabolism, we investigated in anesthetized
rats the effects of diallyldisulfide on the secretion of luteinizing
hormone (LH) from the pituitary gland, which regulates tes-
tosterone production in the testis (12,13).
MATERIALS AND METHODS
Animal care. Male Sprague-Dawley rats (Japan SLC, Shizuoka,
Japan) were housed individually in stainless steel wire-bottom cages
in a room maintained at 22–24°C and ⬃50% relative humidity. The
room was lit from 0700 to 1900 h. Tap water was freely available.
Rats, 4 and 7 wk old, were purchased for use in Experiments 1 and 2,
respectively. In Experiment 1, rats were fed a commercial diet (CE-2,
Japan, Clea, Tokyo, Japan) for 3 d before starting the experiments,
and in Experiment 2, rats were given the commercial diet before
starting the experiments. This study was approved by the Institutional
Animal Care and Use Committee of Kobe Women’s University,
Faculty of Home Economics.
Experiment 1. The experimental diets were normal fat (5 g/100
g fat) diets with three different protein levels (40, 25 or 10 g/100 g
casein), as shown in Table 1. Rats in the control group were fed 40,
25 or 10% casein (control diet), whereas rats in the garlic group were

0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences.

Manuscript received 27 November 2000. Initial review completed 4 January 2001. Revision accepted 11 May 2001.

2150
 GARLIC AFFECTS TESTOSTERONE AND CORTICOSTERONE
TABLE 1
Composition of experimental diets (Experiment 1)
40% casein 25% casein 10% casein
diet diet diet
g/kg
Casein1 400 250 100
Corn oil1 50 50 50
Vitamins2 17 17 17
Minerals3 50 50 50
Cellulose1 40 40 40
Sucrose1 300 300 300
␣-Cornstarch1 143 293 443
Garlic4 — — — contamination of the emulsion layer. The extract was washed twice
Energy density,5
MJ/kg 15.98 15.98 15.98
1 Oriental Yeast, Tokyo, Japan
2 Purchased from Oriental Yeast. Vitamin mixture (mg/kg diet) con-
tained retinyl acetate 17, cholecalciferol 0.0425, all-rac-␣-tocopherol
acetate 85, menadione 88.4, thiamin HCl 20.4, riboflavin 68, pyridoxine
HCl 13.6, vitamin B-12 0.0085, vitamin C 510, D-biotin 0.34, folic acid
3.4, Ca-pantothenate 85, p-aminobenzoic acid 85, nicotinic acid 102,
inositol 102, choline chloride 3400, and cellulose powder 12,419.809.
3 Purchased from Oriental Yeast. Mineral mixture (mg/kg diet) con-
tained CaHPO4 䡠 H2O 7280, KH2PO4 12,860, NaH2PO4 䡠 H2O 4675,
NaCl 2330, Ca-lactate 17,545, Fe-citrate 1590, MgSO4 䡠 H2O 3585,
ZnCO3 55, MnSO4 䡠 4 䡠 5H2O 60, CuSO4 䡠 5H2O 15, and KI 5.
4 Added at 8 g/kg diet as garlic powder to each experimental garlic
diet. Prepared by Riken Chemical (Kyoto, Japan). The composition of
garlic powder was as follows (%): water 5.5, ash 3.2, protein 17.2, fat
0.4, fiber 1.4, and carbohydrate 72.3. The volatile compounds con-
tained a total of 5.05 mg of total diallylsulfide (0.05 mg of monosulfide,
1.0 mg of disulfide, 3.4 mg of trisulfide, 0.6 mg tetrasulfide) per gram of
garlic powder, which were expressed in diallyldisulfide equivalents.
5 Energy values were as follows: 16.70 MJ/kg for starch, soluble
carbohydrates and protein and 37.70 MJ/kg for fat.
fed one of these diets supplemented with 8 g of garlic powder/kg diet
(garlic diet). The garlic powder was prepared from fresh garlic bulb
(Riken Chemical Industry, Kyoto, Japan), which was heat-dried at
60 –70°C, and then ground by a mill. Volatile compounds in the
garlic powder were analyzed by gas chromatography using diallyl-
disulfide as a standard (14,15); their concentrations were determined
as diallyldisulfide equivalents (Table 1). Rats (n ⫽ 39) weighing
80 –90 g were separated into six groups (control groups, 6 rats; garlic
groups, 7 rats) and were fed for 28 d one of the following experimental
diets: 40, 25 or 10% casein diets with or without garlic powder. Each
group was fed the appropriate diet in amounts such that the six groups
consumed an equal amount of metabolizable energy during the ex-
perimental period, and that food consumption of each of the six
groups was approximately equivalent to the maximal amount (food
intake was sufficient for the rats) that rats can consume under these
conditions. At the end of the 28 d, the rats were transferred to
individual metabolic cages and urine and feces were collected sepa-
rately for 1 d. To each urine sample obtained during the collection
was added 1 mL of 6 mol/L HCl solution to prevent its degradation.
After the collection, urinary and fecal nitrogen contents and the
nitrogen content in each experimental diet were determined by the
semimicro Kjeldahl method, and nitrogen intake was calculated on
the basis of the nitrogen content of each experimental diet from the
total amount of food consumed. Urinary creatinine content was
measured using the method of Clark and Thompson (16). Urinary
17-ketosteroid (urinary excretion of total amounts of androsterone,
etiocholanone, dehydroepiandrosterone, 11-ketoandrosterone, 11-
ketoetiocholanone, 11-OH androsterone and 11-OH etiocholanor-
one) content was determined by the Zimmerman reaction (17).
The rats were anesthetized using ␣-chloralose and urethane (18),
which were purchased from Wako Chemical (Osaka, Japan) and
Tokyo Chemical (Tokyo, Japan), respectively. After feeding (at the
2151
end of d 29), rats were anesthetized by intraperitoneal injection of
␣-chrolarose and urethane (75 and 750 mg/kg, respectively). Blood
samples were collected from the abdominal aorta, and plasma was
separated after centrifugation (3000 ⫻ g for 15 min) and stored at
⫺40°C until analyzed. After collection of the blood sample, the liver,
kidney, perirenal adipose tissues and epididymal fat pad were imme-
diately excised, weighed and stored at ⫺40°C for further analyses.
Arginase activity in the liver was determined using the method of
Schimke (19,20). Plasma corticosterone concentration was deter-
mined using the method of Sagara et al. (21), i.e., the plasma sample
was analyzed by HPLC after the extraction of steroids, as described
below. To extract free steroids in the plasma, 10 mL of 0.05%
methanol-chloroform and 0.2 mL of 1 mol/L NaOH were added to 1
mL of plasma sample, and the mixture was shaken gently for 20 min.
Then, 9 mL of the methanol-chloroform layer was removed without
with distilled water (2 mL); 8 mL of the methanol-chloroform layer
was obtained and evaporated to dryness in a rotary evaporator under
continuous suction. The residue was dissolved in absolute methanol.
An aliquot of the solution was injected into a chromatograph [Irica
HPLC system; detection, UV at 245 nm; flow rate, 0.5 mL/min;
column, ODS column RP-18T, 250 ⫻ 4.6 mm; mobile phase, H2O/
methanol/tetrahydrofuran (36:55:9)]. Each rat testis was homoge-
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nized with 1 mL of distilled water and centrifuged at 12,000 ⫻ g at
4°C for 30 min (22). Then the supernatant was separated and steroids
were extracted using the same method as in the case of plasma
corticosterone. After steroid extraction, the testicular testosterone
content was analyzed by HPLC as described above.
In a preliminary experiment concerning the effects of different
cholesterol concentrations in two different fats, testosterone content
in the testis was compared in rats fed high fat diets (21.21 MJ/kg)
containing 30% shortening (0% cholesterol) or lard (0.1% choles-
terol). The composition of the shortening and lard diets was described
previously (9). We examined the effects of garlic supplementation (8
g/kg diet) on testicular testosterone content in rats fed these diets for
28 d. Each group of rats was offered the appropriate diet in amounts
such that the four groups consumed equal metabolizable energy
during the experimental period, and food consumption in all four
groups was approximately equivalent to the maximum (food intake
was sufficient for the rats) that rats can consume under these condi-
tions. Testicular testosterone content was determined by the same
method as in Experiment 1 described above.
Experiment 2. Rats weighing ⬃250 g were anesthetized as de-
scribed above; their rectal temperature was maintained between 36.5
and 37.5°C using a direct-current heating pad. Rats (n ⫽ 6 –7) were
used to evaluate the effects of diallyldisulfide in comparison with rats
that received vehicle alone (9 g/L NaCl solution containing 2%
ethanol and 10% Tween 80). We determined the dose-dependent
response with respect to plasma LH concentration after the admin-
istration of diallyldisulfide. Diallyldisulfide [88.9%; the remaining
compounds were diallylmonosulfide (5.4%) and diallyltrisulfide
(5.3%)] was purchased from Tokyo Chemical. For dose-response
measurements, each rat received 1 mL of the vehicle containing 10
mmol/L (1.46 mg), 20 mmol/L (2.92 mg) or 30 mmol/L (4.28 mg)
diallyldisulfide via injection into the right femoral vein over 1 min.
Blood samples were collected from the abdominal aorta after 30 min.
In a preliminary experiment, we confirmed that plasma LH concen-
trations were maximal 30 min after administration. Accordingly, we
performed the dose-response measurements and determined plasma
LH concentration 30 min after diallyldisulfide administration. Plasma
LH concentration was assayed using an enzyme immunoassay kit (rat
LH enzyme immunoassay system, Amersham Pharmacia, UK).
In another preliminary experiment concerning the effects of nor-
adrenaline on plasma LH concentration, plasma LH concentration in
rats after administration of 5, 10 or 50 mg noradrenaline was exam-
ined as described above.
Statistical analysis. All data are presented as means ⫾ SEM.
Statistical analyses were done using Statistical Package for Social
Sciences (SPSS10.0 for Windows; SPSS, Chicago, IL). In Experi-
ment 1, treatment effects (dietary protein levels and garlic supple-
mentation) were analyzed using two-way ANOVA, and the differ-
ences between means were tested using Duncan’s multiple range
2152 OI ET AL.

TABLE 2
Effects of garlic supplementation on body weight, liver, kidney, testis, epididymal fat pad, and perirenal adipose tissue weights,
and urinary creatinine excretion in rats fed diets with different protein levels for 28 d (Experiment 1)1

40% casein diet 25% casein diet 10% casein diet

Control group Garlic group Control group Garlic group Control group Garlic group ANOVA2

Body weight 235.9 ⫾ 3.1a 242.4 ⫾ 4.0a 242.6 ⫾ 3.0a 238.3 ⫾ 2.9a 198.3 ⫾ 3.0b 193.2 ⫾ 2.5b P
Liver weight 9.07 ⫾ 0.15a 8.80 ⫾ 0.10ab 8.15 ⫾ 0.15bc 8.02 ⫾ 0.44c 5.18 ⫾ 0.23d 5.40 ⫾ 0.39d P
Kidney weight 2.08 ⫾ 0.03a 2.12 ⫾ 0.02a 1.91 ⫾ 0.05a 1.97 ⫾ 0.05a 1.45 ⫾ 0.03b 1.43 ⫾ 0.03b P
Testis weight 2.62 ⫾ 0.08 2.50 ⫾ 0.12 2.58 ⫾ 0.05 2.59 ⫾ 0.05 2.31 ⫾ 0.08 2.34 ⫾ 0.08 NS
Epididymal fat pad weight 3.59 ⫾ 0.44 3.50 ⫾ 0.11 3.86 ⫾ 0.22 3.55 ⫾ 0.19 3.34 ⫾ 0.35 2.94 ⫾ 0.21 NS
Perirenal adipose tissue weight 0.76 ⫾ 0.09 0.76 ⫾ 0.05 0.81 ⫾ 0.06 0.81 ⫾ 0.02 0.71 ⫾ 0.07 0.61 ⫾ 0.06 NS

␮mol/d

Urinary creatinine 96.8 ⫾ 4.5ab 103.9 ⫾ 6.9a 90.3 ⫾ 4.8b 95.5 ⫾ 6.7ab 71.4 ⫾ 5.0c 80.7 ⫾ 2.9bc P

1 Values are means ⫾ SEM, n ⫽ 6 (control) or 7 (garlic) rats. Within a row, values with a superscript not sharing a letter are different, P ⬍ 0.05.
2 Two-way ANOVA: P, significant influence of dietary protein level (P ⱕ 0.05); NS, not significant (P ⬎ 0.05).

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post-hoc test. In Experiment 2, data were analyzed using one-way
ANOVA, and significant differences between means were evaluated
by the Bonferroni post-hoc test. The correlations between the data of
plasma LH concentrations and the administration of diallyldisulfide
or noradrenaline for dose response measurements were tested by
regression analysis. Differences with P ⬍ 0.05 were considered sig-
nificant.
RESULTS
Experiment 1. After 28 d of dietary treatment, no signif-
icant differences due to garlic were found on body, liver,
kidney, testis, perirenal adipose tissue and epididymal fat pad
weights, or urinary creatinine (Table 2). Urinary nitrogen was
significantly lower in the garlic-40% casein diet group than in
the control-40% casein diet group (Table 3). No differences
were observed among groups in fecal nitrogen content
(Table 3). Nitrogen balance was significantly higher in the
garlic-40% casein diet group than in the control-40% casein
diet group, whereas there were no significant differences be-
tween the control diet groups and the garlic diet groups fed 10
and 25% casein diets (Table 3). Similarly, arginase activity in
the liver was significantly higher in the garlic-40% casein diet
group than in the control-40% casein diet group, whereas
there were no significant differences between the control diet
groups and the garlic diet groups fed 10 and 25% casein diets
(Table 4). Plasma corticosterone concentrations in rats fed all
levels of casein were significantly lower in those supplemented
with garlic (Fig. 1). Testosterone contents in the testis of rats
fed either 40 or 25% casein diets were significantly greater in
rats supplemented with garlic, whereas there were no signifi-
cant differences between the control diet group and the garlic
diet group fed the 10% casein diet (Fig. 2). Urinary 17-
ketosteroid levels in rats fed 40% casein diets were signifi-
cantly greater in those consuming garlic (Fig. 3). Furthermore,
urinary 17-ketosteroid excretion in rats in the garlic-40%
casein diet group was significantly higher than that in the
garlic-10% casein diet group (Fig. 3).
In the preliminary experiment, we examined the effects of
different cholesterol levels in shortening and lard diets on
testicular testosterone content. The testicular testosterone lev-
els in lard-fed rats were significantly higher than those in
shortening-fed rats, and garlic supplementation increased lev-
els in rats fed both diets (Fig. 4). Further investigation is
necessary to elucidate the different effects of garlic supplemen-

TABLE 3
Effects of garlic supplementation on urinary nitrogen content, fecal nitrogen content and nitrogen balance
in rats fed diets with different protein levels for 28 d (Experiment 1)1

40% casein diet 25% casein diet 10% casein diet

Control group Garlic group Control group Garlic group Control group Garlic group ANOVA2

mg N/d

Nitrogen intake 926.8 931.8 593.9 595.1 257.8 245.0

Urinary nitrogen 644.3 ⫾ 30.5a 513.6 ⫾ 40.8b 310.4 ⫾ 17.5c 311.7 ⫾ 15.7c 113.8 ⫾ 4.9d 120.5 ⫾ 10.1d P, G, PG
Fecal nitrogen 27.1 ⫾ 3.0 30.0 ⫾ 3.2 27.8 ⫾ 2.9 27.7 ⫾ 1.1 21.9 ⫾ 1.8 17.8 ⫾ 0.9 NS
Nitrogen balance 257.9 ⫾ 30.1b 413.8 ⫾ 50.3a 261.5 ⫾ 17.0b 255.6 ⫾ 15.3b 126.1 ⫾ 4.9c 106.8 ⫾ 9.9c P, G, PG

1 Values are means ⫾ SEM, n ⫽ 6 (control) or 7 (garlic) rats. Within a row, values with a superscript not sharing a letter are different, P ⬍ 0.05.
2 Two-way ANOVA: Significant influence of dietary protein level (P), garlic (G), interaction of protein and garlic (PG), P ⬍ 0.05; NS, not significant
(P ⱖ 0.05).
 GARLIC AFFECTS TESTOSTERONE AND CORTICOSTERONE 2153

TABLE 4
Effects of garlic supplementation on liver weight and arginase activity in rats fed diets with different protein
levels for 28 d (Experiment 1)1

40% casein diet 25% casein diet 10% casein diet

Control group Garlic group Control group Garlic group Control group Garlic group ANOVA2

Arginase3
␮mol/(min 䡠 g liver) 0.241 ⫾ 0.017b 0.305 ⫾ 0.016a 0.207 ⫾ 0.025bc 0.222 ⫾ 0.017b 0.162 ⫾ 0.012c 0.172 ⫾ 0.013c P,G,PG
␮mol/(min 䡠 liver) 1.884 ⫾ 0.153b 2.468 ⫾ 0.160a 1.730 ⫾ 0.061b 1.766 ⫾ 0.111b 0.835 ⫾ 0.068c 0.900 ⫾ 0.056c P,G,PG

1 Values are means ⫾ SEM, n ⫽ 6 (control) or 7 (garlic). Within a row, values with a superscript not sharing a letter are different, P ⬍ 0.05.
2 Two-way ANOVA: Significant influence of dietary protein level (P), garlic (G), interaction of protein and garlic (PG); P ⬍ 0.05.
3 Arginase activity indicates urea synthesis rate in the liver, and is expressed as ␮mol of urea produced per min at 25°C.

tation, in relation to dietary cholesterol, on steroid hormone
production.
Experiment 2. Plasma LH concentrations were signifi-
cantly higher in rats that received 20 or 30 mmol/L diallyl-
disulfide than in those that received vehicle alone (Table 5).
of whole-body protein synthesis and breakdown to be esti-
mated together with amino acid oxidation and the fractional
synthetic rates of mixed muscle protein or of single plasma
proteins (10,23). Protein synthesis rates of tissues (e.g., plasma,
tibialis anterior, soleus or liver) in rats in vivo were measured

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The increase was dose dependent, and there was a positive
correlation (P ⬍ 0.01, r ⫽ 0.552) between plasma LH con-
centration and the dose of diallyldisulfide.
In the preliminary experiment, we examined the effects of
noradrenaline on plasma LH concentration. Noradrenaline
increased plasma LH concentration and its effects on plasma
LH concentrations were dose dependent (P ⬍ 0.001, r
⫽ 0.617) (Table 6).
DISCUSSION
Most of the recent knowledge concerning the regulation of
protein metabolism in humans has been obtained by tracing
protein kinetics in vivo using labeled isotopes of essential or
nonessential amino acids (23). This technique allows the rates
by the flooding dose method, which reduces uncertainty over
the labeling of the tracer amino acid in the precursor pool for
the protein synthesis (24 –26). Broadly speaking, isotopic
methods fall into two groups, i.e., those methods based on
uptake of isotope into protein which yield information about
the rate of protein synthesis and those based on isotope loss
from which the rates of both synthesis and breakdown can be
determined (27). For instance, enzymes must have a fast turn-
over rate so that their concentrations can be rapidly changed
by factors that modulate their rates of synthesis or degradation.
Among the potential factors that play a role in the regulation
of protein metabolism are a number of substrates and hor-
mones. Hormones, by affecting the turnover rates of key pro-
teins, can modulate cell differentiation and growth and direct
the flux of substrates through specific metabolic pathways
(10,23). Our previous papers indicated that the allyl-contain-

FIGURE 1 Effects of garlic supplementation on plasma cortico-

sterone concentration in rats fed diets with different protein levels
(Experiment 1). Values are means ⫾ SEM for 6 (control) or 7 (garlic) rats.
Means with different letters are significantly different, P ⬍ 0.05. The
influences of dietary protein level and garlic supplementation and the
interaction between dietary protein level and garlic supplementation
were significant, P ⬍ 0.05.
FIGURE 2 Effects of garlic supplementation on testicular testos-
terone content in rats fed diets with different protein levels (Experiment
1). Values are means ⫾ SEM for 6 (control) or 7 (garlic) rats. Means with
different letters are significantly different, P ⬍ 0.05. The influence of
garlic supplementation and the interaction between dietary protein level
and garlic supplementation were significant, P ⬍ 0.05.
2154 OI ET AL.
FIGURE 3 Effects of garlic supplementation on urinary excretion
of 17-ketosteroid (urinary excretion of total amounts of androsterone,
etiocholanone, dehydroepiandrosterone, 11-ketoandrosterone, 11-ke-
toetiocholanone, 11-OH androsterone and 11-OH etiocholanorone) in
rats fed diets with different protein levels (Experiment 1). Values are
means ⫾ SEM for 6 (control) or 7 (garlic) rats. Means with different letters
are significantly different, P ⬍ 0.05. The influence of garlic supplemen-
tation and the interaction between dietary protein level and garlic
supplementation were significant, P ⬍ 0.05.
ing polysulfides in garlic are responsible for the enhancement
of noradrenaline and adrenaline secretion and the increased
thermogenesis indicated by the increased UCP content in
IBAT (9). We speculated that garlic may be involved in
hormonal secretion. Thus, garlic administration may affect
FIGURE 4 Effects of garlic supplementation on testicular testos-
terone content in rats fed shortening or lard diet for 28 d. Values are
means ⫾ SEM for 6 (control) or 7 (garlic) rats. Means with different letters
are significantly different, P ⬍ 0.05. Influences of dietary fat source and
garlic supplementation and the interaction between dietary fat source
and garlic supplementation were significant, P ⬍ 0.05.
TABLE 5
Dose response of plasma luteinizing hormone concentration
in rats following diallyldisulfide administration (Experiment 2)1
␮g/L plasma
Vehicle alone2 3.40 ⫾ 0.08c
Diallyldisulfide,2 mmol/L
10 4.77 ⫾ 0.62bc
20 6.09 ⫾ 0.57ab
30 7.52 ⫾ 0.58a
1 Values are means ⫾ SEM, n ⫽ 6 (vehicle alone) or 7 (10, 20 or 30
mmol/L of diallyldisulfide). Values not sharing a superscript are differ-
ent, P ⬍ 0.05.
2 Each rat received 1 mL of the vehicle (9 g/L NaCl solution con-
taining 2% ethanol and 10% Tween 80) containing 10 mmol/L (1.46
mg), 20 mmol/L (2.92 mg) or 30 mmol/L (4.28 mg) diallyldisulfide,
injected into the right femoral vein over 1 min. All of the blood samples
from each rat were collected from the abdominal aorta after 30 min.
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whole-body protein metabolism due to hormonal regulation by
stimulating hormone secretion, and dietary supplementation
of garlic may affect protein metabolism by enhancing protein
anabolism. The present study was conducted to investigate the
effects of garlic on protein metabolism, particularly to deter-
mine the effects of garlic supplementation on the hormonal
regulation of protein metabolism by measuring the levels of
steroid hormones, i.e., testosterone and corticosterone. The
following hormones have been suggested to affect protein
metabolism: insulin, growth hormone, insulin-like growth fac-
tor I, adrenaline, androgen, estrogen, progesterone, glucagon,
glucocorticoid and thyroid hormone (10). On the basis of their
net effects on protein balance (protein synthesis minus protein
breakdown) in the entire body, hormones can be classified into
two groups as follows: those having a prevalent anabolic ac-
tion, i.e., insulin, growth hormone, insulin-like growth factor
I, adrenaline and androgen (testosterone), and those with a
prevalent catabolic action, i.e., glucocorticoids (corticoste-
rone), glucagon and thyroid hormone. Therefore, in the
present study, we investigated the effects of garlic administra-
tion on testosterone as a protein anabolic hormone and cor-
ticosterone as a protein catabolic hormone.
In Experiment 1, the effects of garlic powder supplementa-
tion on protein metabolism in rats fed the experimental diet
containing different casein levels (40, 25 or 10% casein diet)
TABLE 6
Dose response of plasma luteinizing hormone concentration
in rats following noradrenaline administration (Experiment 2)1
␮g/L plasma
Vehicle alone2 3.92 ⫾ 0.23c
Noradrenaline2
5 ng 15.16 ⫾ 0.74b
10 ng 18.39 ⫾ 0.44ab
50 ng 21.80 ⫾ 2.19a
1 Values are means ⫾ SEM, n ⫽ 6 (vehicle alone) or 7 (noradrenaline).
Values not sharing a superscript are different, P ⬍ 0.05.
2 Each rat received 1 mL of the vehicle (9 g/L NaCl solution con-
taining 2% ethanol and 10% Tween 80) containing 5, 10 or 50 ng
noradrenaline, injected into the right femoral vein over 1 min. All of the
blood samples for each rat were collected from the abdominal aorta 30
min after administration.
 GARLIC AFFECTS TESTOSTERONE AND CORTICOSTERONE
were investigated. Testicular testosterone content, urinary 17-
ketosteroid content, arginase activity in the liver and nitrogen
balance were significantly increased in rats after garlic supple-
mentation to the 40% casein diet, whereas plasma corticoste-
rone concentration was significantly decreased in rats after
garlic supplementation to the 40 or 25% casein diet. Based on
urinary excretion of creatinine data, body muscle mass was not
affected by garlic supplementation. However, nitrogen balance
data suggested that nitrogen retention in the body was en-
hanced by garlic supplementation in rats fed a high protein
diet. Similarly, hepatic arginase activity data suggested that
protein synthesis in the liver was enhanced by garlic supple-
mentation in rats fed a high protein diet. Urinary 17-keto-
steroid is an index of steroid hormone secretion, which is
derived almost completely from testosterone secretion in the
whole body (i.e., an index of testosterone secretion in testis).
These results suggest that protein anabolism occurs in rats fed
the high protein diet supplemented with garlic. Concerning
the effects of garlic on protein metabolism, the different re-
sponses to garlic supplementation in rats fed normal-fat diets
with different protein levels suggest that protein anabolic
effects were induced by the high protein diet (40% casein
diet), but not by the low protein diet (10% casein diet). The
present study suggests that to induce the protein anabolic
effect of garlic supplementation, the protein content in the
diet should be high. Therefore, our findings suggest that pro-
tein anabolic effects were induced to a greater extent by garlic
supplementation in rats fed the high protein experimental
diet.
Steroid hormones are produced from cholesterol in mam-
mals. The experimental diets in the present study were nor-
mal-fat diets containing 5% corn oil, which is cholesterol free.
The testosterone contents in rats fed the 40, 25 or 10% casein
diet were not significantly different, whereas the testosterone
contents in rats fed the 40% casein diet were significantly
increased by garlic supplementation. We speculate that testic-
ular testosterone was derived from the de novo synthesis of
cholesterol in the body. Therefore, our data indicate that
garlic supplementation enhanced testosterone production in
the testis.
LH is a glycoprotein with a molecular weight of
⬃36,000; it is comprised of two subunits (␣ and ␤). LH is
termed gonadotrophin and is secreted by basophilic cells of
the anterior pituitary gland, called gonadotrophs. It has
been reported that LH stimulates Leydig cells in the testis
to produce testosterone (12,13,28). In Experiment 2, to
confirm the protein anabolic effects of garlic, the effects of
diallyldisulfide on the secretion of LH from the pituitary
gland, which regulates testosterone production in the testis,
were investigated in anesthetized rats. In this experiment,
plasma LH concentration was directly affected by diallyld-
isulfides and the intravenous administration of diallyldisul-
fide corresponded to garlic absorption in blood after oral
consumption. The dose of diallyldisulfide (10 mmol/L, 1.46
mg) corresponded to approximately twice the total average
amount of garlic consumed per day per rat in Experiment 1;
thus, it is considered to be equivalent to the physiologic
level of garlic in rats. Therefore, we evaluated the effects of
diallyldisulfide on plasma LH concentration to determine
specifically the dose-dependent response of plasma LH con-
centration after diallyldisulfide administration. Diallyldisulfide in-
creased plasma LH concentration, and plasma LH concentra-
tion was affected by diallyldisulfide in a dose-dependent
manner. These results suggest that garlic administration in-
creases testosterone production in the testis due to the en-
2155
hancement of LH secretion from the pituitary gland. Previ-
ously, we reported that allyl-containing sulfides in garlic
increased noradrenaline and adrenaline secretion levels (7–9).
Noradrenaline and adrenaline, which are involved in the
secretion of various hormones, play important roles in stimu-
lating hormone secretion. Our results (Table 6) suggest that
increasing noradrenaline secretion via stimulation by allyl-
containing sulfides in garlic enhances LH secretion from the
pituitary gland. Therefore, we contend that allyl-containing
sulfides in garlic are responsible for the enhancement of LH
secretion via stimulation of the pituitary gland by noradrena-
line. Garlic supplementation likely increases testicular testos-
terone content due to the stimulation of LH secretion from the
pituitary gland by the increased plasma noradrenaline concen-
tration. The present study suggests that garlic supplementation
enhances protein anabolism and suppresses protein catabolism
due to hormonal regulation by the stimulation of steroid
hormones, leading to greater testis testosterone content and
lower plasma corticosterone concentration in rats fed a high
protein diet.
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