garkic increases testosterone

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

KEY WORDS:

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garlic

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diallyldisulfide

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testosterone

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, anticoagulative, 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 diallyldisulfide, a major volatile sulfur-containing compound in garlic, enhanced triglyceride catabolism and growth of interscapular brown adipose tissue (IBAT)2 by increasing noradrenaline secretion in rats (7,8). Recently, we reported that allyl-containing 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 corticosterone, affects protein catabolism in rats (10). The present study was conducted to investigate the effects of garlic supple-

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corticosterone

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rats

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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 corticosterone concentrations were significantly lower in rats fed 40 and 25% casein diets with garlic powder than in those 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.

mentation on protein metabolism in rats. In particular, we wanted to determine whether garlic supplementation stimulates 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. Furthermore, to better understand the effects of garlic supplementation 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 testosterone 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

1 To whom correspondence should be addressed.

E-mail: oi@suma.kobe-wu.ac.jp.

2 Abbreviations used: IBAT, interscapular brown adipose tissue; LH, luteinizing hormone; UCP, uncoupling protein.

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.

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TABLE 1 Composition of experimental diets (Experiment 1) 40% casein diet

25% casein diet

10% casein diet

g/kg Casein1 Corn oil1 Vitamins2 Minerals3 Cellulose1 Sucrose1 ␣-Cornstarch1 Garlic4 Energy density,5 MJ/kg

400 50 17 50 40 300 143 — 15.98

250 50 17 50 40 300 293 — 15.98

100 50 17 50 40 300 443 — 15.98

1 Oriental Yeast, Tokyo, Japan 2 Purchased from Oriental Yeast. Vitamin mixture (mg/kg diet) con-

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 diallyldisulfide 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 experimental 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 separately 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, 11ketoetiocholanone, 11-OH androsterone and 11-OH etiocholanorone) 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

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 immediately 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 determined 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 contamination of the emulsion layer. The extract was washed twice 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 homogenized 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% cholesterol). 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 conditions. Testicular testosterone content was determined by the same method as in Experiment 1 described above.

Experiment 2. Rats weighing ⬃250 g were anesthetized as described 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 administration 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 concentrations 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 noradrenaline on plasma LH concentration, plasma LH concentration in rats after administration of 5, 10 or 50 mg noradrenaline was examined 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 Experiment 1, treatment effects (dietary protein levels and garlic supplementation) were analyzed using two-way ANOVA, and the differences between means were tested using Duncan’s multiple range

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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) contained 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 contained 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.

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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 Control group

25% casein diet

Garlic group

Control group

10% casein diet

Garlic group

Control group

Garlic group

ANOVA2

238.3 ⫾ 2.9a 8.02 ⫾ 0.44c 1.97 ⫾ 0.05a 2.59 ⫾ 0.05 3.55 ⫾ 0.19 0.81 ⫾ 0.02

198.3 ⫾ 3.0b 5.18 ⫾ 0.23d 1.45 ⫾ 0.03b 2.31 ⫾ 0.08 3.34 ⫾ 0.35 0.71 ⫾ 0.07

193.2 ⫾ 2.5b 5.40 ⫾ 0.39d 1.43 ⫾ 0.03b 2.34 ⫾ 0.08 2.94 ⫾ 0.21 0.61 ⫾ 0.06

P P P NS NS NS

71.4 ⫾ 5.0c

80.7 ⫾ 2.9bc

P

g Body weight Liver weight Kidney weight Testis weight Epididymal fat pad weight Perirenal adipose tissue weight

235.9 ⫾ 3.1a 9.07 ⫾ 0.15a 2.08 ⫾ 0.03a 2.62 ⫾ 0.08 3.59 ⫾ 0.44 0.76 ⫾ 0.09

242.4 ⫾ 4.0a 8.80 ⫾ 0.10ab 2.12 ⫾ 0.02a 2.50 ⫾ 0.12 3.50 ⫾ 0.11 0.76 ⫾ 0.05

242.6 ⫾ 3.0a 8.15 ⫾ 0.15bc 1.91 ⫾ 0.05a 2.58 ⫾ 0.05 3.86 ⫾ 0.22 0.81 ⫾ 0.06

␮mol/d Urinary creatinine

96.8 ⫾ 4.5ab

103.9 ⫾ 6.9a

90.3 ⫾ 4.8b

95.5 ⫾ 6.7ab

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 significant.

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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).

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 significant differences between the control diet group and the garlic diet group fed the 10% casein diet (Fig. 2). Urinary 17ketosteroid levels in rats fed 40% casein diets were significantly 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 levels in lard-fed rats were significantly higher than those in shortening-fed rats, and garlic supplementation increased levels in rats fed both diets (Fig. 4). Further investigation is necessary to elucidate the different effects of garlic supplemen-

RESULTS Experiment 1. After 28 d of dietary treatment, no significant 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 between 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

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 Control group

Garlic group

25% casein diet Control group

Garlic group

10% casein diet Control group

Garlic group

ANOVA2

257.8 113.8 ⫾ 4.9d 21.9 ⫾ 1.8 126.1 ⫾ 4.9c

245.0 120.5 ⫾ 10.1d 17.8 ⫾ 0.9 106.8 ⫾ 9.9c

P, G, PG NS P, G, PG

mg N/d Nitrogen intake Urinary nitrogen Fecal nitrogen Nitrogen balance

926.8 644.3 ⫾ 30.5a 27.1 ⫾ 3.0 257.9 ⫾ 30.1b

931.8 513.6 ⫾ 40.8b 30.0 ⫾ 3.2 413.8 ⫾ 50.3a

593.9 310.4 ⫾ 17.5c 27.8 ⫾ 2.9 261.5 ⫾ 17.0b

595.1 311.7 ⫾ 15.7c 27.7 ⫾ 1.1 255.6 ⫾ 15.3b

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

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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

Arginase3 ␮mol/(min 䡠 g liver) ␮mol/(min 䡠 liver)

25% casein diet

10% casein diet

Control group

Garlic group

Control group

Garlic group

Control group

Garlic group

ANOVA2

0.241 ⫾ 0.017b 1.884 ⫾ 0.153b

0.305 ⫾ 0.016a 2.468 ⫾ 0.160a

0.207 ⫾ 0.025bc 1.730 ⫾ 0.061b

0.222 ⫾ 0.017b 1.766 ⫾ 0.111b

0.162 ⫾ 0.012c 0.835 ⫾ 0.068c

0.172 ⫾ 0.013c 0.900 ⫾ 0.056c

P,G,PG 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.

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

FIGURE 1 Effects of garlic supplementation on plasma corticosterone 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.

of whole-body protein synthesis and breakdown to be estimated 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 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 turnover 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 hormones. Hormones, by affecting the turnover rates of key proteins, 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-

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tation, in relation to dietary cholesterol, on steroid hormone production.

Experiment 2. Plasma LH concentrations were significantly higher in rats that received 20 or 30 mmol/L diallyldisulfide than in those that received vehicle alone (Table 5).

The increase was dose dependent, and there was a positive correlation (P ⬍ 0.01, r ⫽ 0.552) between plasma LH concentration 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).

FIGURE 2 Effects of garlic supplementation on testicular testosterone 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.

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TABLE 5 Dose response of plasma luteinizing hormone concentration in rats following diallyldisulfide administration (Experiment 2)1 ␮g/L plasma Vehicle alone2 Diallyldisulfide,2 mmol/L 10 20 30

3.40 ⫾ 0.08c 4.77 ⫾ 0.62bc 6.09 ⫾ 0.57ab 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 different, P ⬍ 0.05.

2 Each rat received 1 mL of the vehicle (9 g/L NaCl solution containing 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.

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

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FIGURE 3 Effects of garlic supplementation on urinary excretion of 17-ketosteroid (urinary excretion of total amounts of androsterone, etiocholanone, dehydroepiandrosterone, 11-ketoandrosterone, 11-ketoetiocholanone, 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 supplementation and the interaction between dietary protein level and garlic supplementation were significant, P ⬍ 0.05.

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 determine 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 factor 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 action, i.e., insulin, growth hormone, insulin-like growth factor I, adrenaline and androgen (testosterone), and those with a prevalent catabolic action, i.e., glucocorticoids (corticosterone), glucagon and thyroid hormone. Therefore, in the present study, we investigated the effects of garlic administration on testosterone as a protein anabolic hormone and corticosterone as a protein catabolic hormone.

In Experiment 1, the effects of garlic powder supplementation 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 Noradrenaline2 5 ng 10 ng 50 ng

FIGURE 4 Effects of garlic supplementation on testicular testosterone 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.

3.92 ⫾ 0.23c 15.16 ⫾ 0.74b 18.39 ⫾ 0.44ab 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 containing 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

hancement of LH secretion from the pituitary gland. Previously, 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 stimulating hormone secretion. Our results (Table 6) suggest that increasing noradrenaline secretion via stimulation by allylcontaining 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 noradrenaline. Garlic supplementation likely increases testicular testosterone content due to the stimulation of LH secretion from the pituitary gland by the increased plasma noradrenaline concentration. 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.

LITERATURE CITED

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were investigated. Testicular testosterone content, urinary 17ketosteroid content, arginase activity in the liver and nitrogen balance were significantly increased in rats after garlic supplementation to the 40% casein diet, whereas plasma corticosterone 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 enhanced 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 supplementation in rats fed a high protein diet. Urinary 17-ketosteroid 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 responses 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 protein 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 mammals. The experimental diets in the present study were normal-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 testicular 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 diallyldisulfides and the intravenous administration of diallyldisulfide 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 concentration after diallyldisulfide administration. Diallyldisulfide increased plasma LH concentration, and plasma LH concentration was affected by diallyldisulfide in a dose-dependent manner. These results suggest that garlic administration increases testosterone production in the testis due to the en-

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