- Email: [email protected]
NDmcr-cp
Life Sciences 80 (2007) 522 – 529 www.elsevier.com/locate/lifescie
Astaxanthin ameliorates features of metabolic syndrome in SHR/NDmcr-cp Ghazi Hussein a,b,⁎, Takako Nakagawa c , Hirozo Goto d , Yutaka Shimada d , Kinzo Matsumoto b , Ushio Sankawa a , Hiroshi Watanabe a a
d
International Research Center for Traditional Medicine, Toyama, Toyama Prefecture 939-8224, Japan b Division of Medicinal Pharmacology, Institute of Natural Medicine, Japan c Department of Kampo Diagnostics, Institute of Natural Medicine, Japan Department of Japanese Oriental Medicine, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan Received 29 May 2006; accepted 28 September 2006
Abstract Glucose and lipid metabolic parameters play crucial roles in metabolic syndrome and its major feature of insulin resistance. This study was designed to investigate whether dietary astaxanthin oil (ASX-O) has potential effects on metabolic syndrome features in an SHR/NDmcr-cp (cp/ cp) rat model. Oral administration of ASX (50 mg/kg/day) for 22 weeks induced a significant reduction in arterial blood pressure in SHRcp. It also significantly reduced the fasting blood glucose level, homeostasis index of insulin resistance (HOMA-IR), and improved insulin sensitivity. The results also showed an improved adiponectin level, a significant increase in high-density lipoprotein cholesterol, a significant decrease in plasma levels of triglycerides, and non-esterified fatty acids. Additionally, ASX showed significant effects on the white adipose tissue by decreasing the size of the fat cells. These results suggest that ASX ameliorates insulin resistance by mechanisms involving the increase of glucose uptake, and by modulating the level of circulating lipid metabolites and adiponectin. © 2006 Elsevier Inc. All rights reserved. Keywords: Astaxanthin; Metabolic syndrome; Blood pressure; Insulin resistance; Lipids; Adiponectin; SHR/NDmcr-cp
Introduction Metabolic syndrome (MetS) is a worldwide-defined (Alberti et al., 2005; WHO, 1999) lifestyle disease that is described as a clustering of multiple metabolic abnormalities, and cardiovascular risk factors including hypertension, obesity, hyperlipidemia, and insulin resistance (InsR) (Eckel et al., 2005). InsR is considered as the central component of this cluster, and is described as a condition whereby there is resistance to insulinmediated glucose uptake by cells. Glucose toxicity (chronic hyperglycemia), and chronically elevated levels of free fatty acids are major metabolic features of MetS. Recently, MetS has become a focus of a number of scientific studies aiming at its prevention, and intervention (Moller, 2001). Current conven⁎ Corresponding author. Division of Medicinal Pharmacology, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Tel.: +81 76 434 7611; fax: +81 76 434 5056. E-mail address: [email protected] (G. Hussein). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.09.041
tional treatment of MetS may include one or more pharmacological therapies, mainly statins (3-hydroxy-3-methylglutaryl coenzyme A inhibitors), angiotensin-converting enzyme inhibitors, fibrates, and thiazolidinediones (Scott, 2003). Diet and lifestyle changes are considered crucial factors in the prevention and treatment. So far, very few reports have described natural products and medicines as candidates against MetS (Shepherd et al., 2005; Xu et al., 2005) and InsR (Mertz, 1993; Nakaya et al., 2000; Peth et al., 2000). Astaxanthin (ASX) is a powerful antioxidant, xanthophyll carotenoid, commonly found in seafood, and predominantly produced by microalga Haematococcus pluvialis Flotow (Lorenz and Cysewski, 2000), which can accumulate more than 30 g of ASX per kg dry biomass (Guerin et al., 2003). We have reported the antihypertensive effects (Hussein et al., 2005b,a, 2006) and action mechanisms (Hussein et al., 2005a) of a dietary carotenoid astaxanthin (ASX-O) in SHR. In this study, we investigated the effects of ASX-O on MetS in SHR/NDmcr-cp (cp/cp), a spontaneously hypertensive rat model of MetS (referred to as
G. Hussein et al. / Life Sciences 80 (2007) 522–529
523
Table 2 The effect of ASX-O administration on body weight in treated animals Treatment period (week)
0 3 6 9 12 15 18 21
Body weight (g) Wistar-OL
SHRcp-OL
SHRcp-ASX
153 ± 1 245 ± 2 317 ± 2 356 ± 3 387 ± 3 402 ± 3 422 ± 3 411 ± 3
149 ± 3 243 ± 7 335 ± 1 405 ± 2 465 ± 3 503 ± 3 539 ± 4 546 ± 3
150 ± 4 244 ± 2 337 ± 2 406 ± 2 463 ± 3 501 ± 2 531 ± 3 533 ± 3
Each value represents the mean ± S.E.M. (n = 6). Fig. 1. Effects of oral administration of ASX-O on systolic blood pressure in SHRcp. The animals received a control vehicle [OL (1 ml/kg/day), open circles], or ASX-O [ASX (50 mg/kg/day), closed circles] for an 18-week treatment period. Each data point represents the mean ± S.E.M. of 6 rats per group. ⁎p b 0.05; ⁎⁎p b 0.01 vs. the control group (t-test).
All the reagents used in these experiments were of analytical grade as follows: Pentobarbital sodium (Tokyo Chemical Industry, Tokyo, Japan), insulin (Novolin R 100, Novo Nordisk, Tokyo), heparin (Novo-Heparin, Mochida, Tokyo), and sodium citrate (Fuso, Osaka, Japan).
on: 07:30−19:30). The animals were housed for at least 1 week before the experiments, and fed a normal diet (Lab MR, NOSAN, Yokohama, Japan) and water ad libitum. Body weight and food intake (FI) were measured daily during the experimental period. ASX-O, composed of 4.5–5.5% (weight per weight) of ASX in an edible oil base, was obtained from Fuji Chemical (Fuji Chemical Industry Co., Ltd, Toyama, Japan), dissolved, and diluted in olive oil (OL) (Wako Pure Chemicals, Osaka, Japan). Administered doses were calculated on the basis of the amount of ASX in the batches of dietary ASX-O. The animals were divided into three groups, a normal Wistar group and two SHRcp groups (6 rats/ group), and were treated once daily for 22 weeks. The ASX-Otreated SHRcp group was administered daily ASX [50 mg (83.8 mmol)/kg, intubation by p.o.], which was measured as 0.1 ml of the stock/100 g bodyweight and diluted, for convenience and acceptability, by an equivalent volume of olive oil prior to administration. The other SHRcp-group and the Wistar group were adopted as control groups and were similarly treated with OL (1 ml/kg/day). All experimental procedures were performed in accordance with the standards established by the ‘Guide for the Care and Use of Laboratory Animals at University of Toyama’.
Animals and drug treatment
Measurement of blood pressure, and heart rate
Male SHR/NDmrc-cp (cp/cp) (designated as SHRcp), and standard Wistar (Wistar/ST) (6 weeks old) rats were used in this study. The animals were obtained from colonies of specific pathogen-free rats maintained by Japan SLC (Shizuoka, Japan). Housing conditions were thermostatically maintained at 24 ± 1 °C with constant humidity (60%) and a 12 h-light/dark cycle (light
Arterial blood pressure and heart rate were determined by a tail cuff system, as previously reported (Hussein et al., 2005b). Briefly, conscious rats were lightly supported in a holder made of cloth mesh and maintained at 37 ± 1 °C (Model THC-1 Digital Thermo, Softron, Tokyo, Japan). Blood pressure from the tail artery was indirectly measured using tail-cuff apparatus (BP-98, Softron), which was controlled with a personal computer. Values are presented as the average of three independent measurements.
SHRcp). A 22-week administration period of ASX at doses of 5 and 50 mg/kg/day, p.o., was examined in SHRcp. In this study, significant changes in the features of MetS were produced by the dose of 50 mg/kg. The effects of ASX-O on blood pressure, blood glucose, blood insulin, insulin sensitivity, and blood lipids were investigated. To the best of our knowledge, this is the first study on the effects of ASX, and carotenoids in general, on MetS. Materials and methods Chemicals
Table 1 Effects of ASX-O on blood pressure and heart rate in SHRcp after treatment for 18 weeks
Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean blood pressure (mmHg) Heart rate (beat/min)
Control (OL)
ASX-O
208 ± 3 179 ± 3 188 ± 3 396 ± 25
189 ± 5⁎ 156 ± 5⁎⁎ 167 ± 1⁎⁎ 374 ± 29
Each value represents the mean ± S.E.M. (n = 6). ⁎p b 0.05; ⁎⁎p b 0.01 vs. the control group (t-test).
Measurement of blood cell counts Blood from the heart of each sacrificed SHRcp was separately collected into heparinized syringes containing 5% heparin, and 2% sodium citrate, and was instantly and gently mixed. A small portion of the blood was used for the cell count, which was determined by a cell counter (Celltac α, Nihon Kohden, Tokyo). The other portion of blood was immediately centrifuged at 3000 g
524
G. Hussein et al. / Life Sciences 80 (2007) 522–529
Table 3 Blood cell counts in SHRcp after treatment with ASX-O for 22 weeks Group
WBC (×102/μl)
RBC (×104/μl)
HGB (g/dl)
HCT (%)
MCV (f l)
MCH (pg)
MCHC (g/dl)
PLT (×104/μl)
Control ASX-O
59.3 ± 1.5 41.0 ± 2.3⁎
950.0 ± 3.1 943.8 ± 4.2
14.9 ± 0.3 15.0 ± 0.2
46.9 ± 0.9 47.4 ± 0.5
49.2 ± 0.6 49.5 ± 0.5
15.6 ± 0.2 15.7 ± 0.2
31.7 ± 0.1 31.7 ± 0.2
72.2 ± 5.3 72.5 ± 0.9
Blood cell count was performed in SHR treated with the control OL (1 ml/kg/day) and ASX (50 mg/kg/day) for 22 weeks. WBC, white blood cell count; RBC, red BC; HGB, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, MC hemoglobin; MCHC, MCH concentration; PLT, platelet count. Each value represents the mean ± S.E.M. (n = 6). ⁎p b 0.01 vs. the control group (t-test).
for 15 min (Kubota 8700, Kubota, Tokyo), and the plasma was separated and used for analysis of the metabolic parameters. Measurement of blood glucose level All rats were fasted overnight with free access to water, and then anesthetized by ethyl ether inhalation. Blood glucose measurement was carried out at 0, 6, 12, and 18 weeks of treatment. Blood was withdrawn from the tail, and instantly tested using a blood glucose-measuring device Antisense III (Horiba, Tokyo, Japan).
Blood sample assays Blood insulin and adiponectin levels were measured using enzyme-linked immunosorbent assay kits (Morinaga Institute of Biological Science, Yokohama, Japan; and Otsuka Pharmaceuticals, Tokyo, Japan, respectively). Blood high-density lipoprotein (HDL)-cholesterol, free fatty acids (FFA), and triglyceride (TG) levels were determined by the HDL-cholesterol E, nonesterified fatty acid (NEFA) C-test, and TG L-type test (Wako, Osaka, Japan), respectively. Histological study
Insulin tolerance test
This model, which incorporates measures of both fasting plasma concentrations of glucose and insulin, was used to calculate the index of InsR as [insulin (μU/ml) × glucose (mM)/ 22.5] (Matthews et al., 1985).
After sacrifice of the animals, small pieces of retroperitoneal white adipose tissue were removed immediately, rinsed with saline and fixed with formalin (10%). Specimens from each rat were embedded in paraffin and sections were stained with hematoxylin and eosin. The specimens and slides were prepared by the Special Reference Laboratories (SRL Tokyo Medical Inc., Tokyo). To examine the size of white adipocytes, the number of adipocytes was counted in limited areas (capture size, 640X480 pixels, equivalent to 12,288 μm2) of each stained specimen. The mean value of the adipocyte count was designated as an index of the cell size; as a larger number means a smaller size. In this study, multilocular cells were excluded and fused cells were counted as a one cell. Examination and photography of the slides was carried out using a light microscopy system (Olympus Provis AX80,
Fig. 2. Fasting blood glucose level in the ASX-treated SHRcp group (closed circles), and the control OL-treated group: SHRcp (open circles), and Wistar (open triangles) rats. Data represent the mean ± S.E.M. (n = 5–6). ⁎p b 0.001 vs. the SHRcp control (t-test).
Fig. 3. Blood glucose level in the ASX-treated SHRcp group (closed circles), and the control OL-treated group: SHRcp (open circles) and Wistar (open triangles) rats, at the insulin tolerance test. Data are the mean ± S.E.M. (n = 5–6). ⁎p b 0.05 vs. the control (t-test).
Tested rats were fasted as above and then injected with insulin 1 ml/kg body weight (equivalent to 0.5 U/kg), i.p. After the injection, blood was obtained from the tail into separate heparinized syringes at intervals of 0 min, 30, 60, and 120 min. Each withdrawn blood sample was immediately tested for blood glucose, as mentioned above. Homeostasis model assessment of insulin resistance (HOMA-IR)
G. Hussein et al. / Life Sciences 80 (2007) 522–529
525
Table 4 Time-course of the effects of ASX-O on glucose metabolic parameters, measured from fasting blood plasma of SHRcp Metabolic parameters
Treatment (week)
Wistar-OL
SHRcp-OL
SHRcp-ASX
Blood glucose (mg/dl)
0 6 18 0 6 18 0 6 18 0 6 18
137.0 ± 15.1 95.3 ± 2.4 128.5 ± 2.3 0.04 ± 0.00 0.09 ± 0.02 0.19 ± 0.03 0.28 ± 0.04 0.52 ± 0.10 1.18 ± 0.09 4.81 ± 0.28 4.44 ± 0.59 4.87 ± 0.29
138.3 ± 4.8 91.7 ± 4.3 154.7 ± 0.9 0.03 ± 0.01 1.47 ± 0.07 1.78 ± 0.04 0.23 ± 0.09 7.88 ± 0.15 16.15 ± 0.30 7.38 ± 0.27 5.91 ± 0.16 5.18 ± 0.15
135.0 ± 2.1 89.3 ± 8.2 123.7 ± 0.3⁎ 0.28 ± 0.03 1.61 ± 0.06 1.63 ± 0.07 2.23 ± 0.15 8.39 ± 0.50 11.87 ± 0.50⁎ 7.08 ± 0.43 8.21 ± 0.14⁎ 5.84 ± 0.29
Blood insulin (ng/ml) HOMA-IR
Adiponectin (μg/ml)
HOMA-IR, homeostasis model assessment of insulin resistance. Each value represents the mean ± S.E.M. (n = 4). ⁎p b 0.01 vs. the SHRcp control (t-test).
Olympus Optical Co. Ltd., Tokyo). Calibration was carried out using a standard area of (1/20 × 1/20 mm) (Burker–Turk haemocytometer, JIS No. E3871, Kayagaki Irika Kogyo Co., LTD., Tokyo). Histological images were measured and analyzed blindly by two investigators using the UTHSCSA Image Tool for Windows, version 3.00 (San Antonio, Texas, USA). Statistical analysis Statistical significance was determined by Student's t-test or Mann–Whitney Rank Sum Test for unpaired observations. Oneway analysis of variance (ANOVA) was performed for multiple comparisons between groups. Differences with p b 0.05 were considered statistically significant. Results Effects of ASX-O on blood pressure In this study, ASX-O showed a significant blood pressurelowering effect on arterial blood pressure, as represented by the effect on systolic blood pressure depicted in Fig. 1 and summarized in Table 1. Throughout the treatment period, the daily administered ASX-O showed no significant or consistent effect on the heart rate. The body weight of ASX-O-treated SHRcp did not significantly change, compared to the SHRcp-OL control group (Table 2). However, the SHRcp groups showed significantly higher body weight than the normal Wistar group, starting from 11 weeks of age ( p b 0.01), and exceeding a 100 g difference from 22 weeks of age ( p b 0.001) (at the age of 25 weeks: Wistar, 420 ± 4 g; SHRcp, 534 ± 4 g). Similarly, no significant differences in daily FI were found among SHRcp groups, which also showed a higher FI than the Wistar group throughout the study (at 25 weeks: Wistar, 19 ± 0.4 g; SHRcp, 21 ± 0.7 g).
Fig. 4. High-density lipoprotein (HDL)-cholesterol level in ASX-O- and control-treated SHRcp, ex vivo. Data are the mean ± S.E.M. (n = 4−5). ⁎p b 0.01 vs. the control (t-test).
except for the white blood cell (WBC) count, which was significantly decreased (Table 3). Effect of ASX-O on blood glucose level The ASX group showed a lower blood glucose level than the corresponding OL group (123.7 ± 0.3 and 154.7 ± 0.9 mg/dl, respectively). The effect was significant and comparable to the normal level in the Wistar group in the 18th week of treatment (Fig. 2). Effect of ASX-O on InsR using insulin tolerance test In insulin tolerance tests, 60 min after the insulin injection, the blood glucose level significantly dropped in the ASX group compared to the OL group. This effect was also maintained at 120 min (Fig. 3), indicating improved insulin sensitivity in SHRcp in the ASX-treated group compared to the control group.
Blood cell counts The 22-week treatment of SHRcp with ASX (50 mg/kg) did not change the blood cell count indices compared to the control,
Fig. 5. Non-esterified fatty acids (NEFA) level in ASX-O- and control-treated animal groups, ex vivo. Data represent the mean ± S.E.M. (n = 4−5). ⁎p b 0.001 vs. the control (t-test).
526
G. Hussein et al. / Life Sciences 80 (2007) 522–529
HDL-cholesterol level, compared to the control (77.77 ± 2.52 and 63.87 ± 2.32 mg/dl, respectively) (Fig. 4). It also exhibited a significantly lower level of NEFA compared to the corresponding control (0.71 ± 0.04 and 1.07 ± 0.07 mEq/l, respectively) (Fig. 5). The ASX-O-treated group also exhibited a significant decrease in TG level compared to the OL group of SHRcp (360.8 ± 24.2 and 467.1 ± 11.8 mg/dl, respectively) (Fig. 6). Effect of ASX-O on adipose tissue
Fig. 6. Triglyceride (TG) level in ASX-O- and control-treated animals, ex vivo. Data represent the mean ± S.E.M. (n = 4). ⁎p b 0.01 vs. the control (t-test).
Time-course of the effects of ASX-O on glucose metabolic parameters
In microscopic studies (Fig. 7), white adipose tissue of the ASX-treated group showed large numbers and aggregates of small fat cells, clearly distinguished as discrete clusters or patches of cells. After treatment with ASX-O for 22 weeks in SHRcp, the number of fat cells was significantly higher in the ASX group than the control [SHRcp-ASX group: 184 ± 3; SHRcp-OL: 140 ± 3 cells/12288 μm2 (mean ± S.E.M.), n = 5–6, p b 0.01, t-test]. Discussion
During a 0–18 week-treatment period, fasting blood plasma of SHRcp showed lower levels of blood glucose and blood insulin in the ASX group compared to the control OL group. The effect was significant on blood glucose after 18-week administration (Table 4). Plasma adiponectin level was higher in the ASX group than that in the control group, with a significant level in the 6th week (8.2 ± 0.1 and 5.9 ± 0.2 g/ml, respectively). The ASX-O-treated group exhibited lower plasma levels of NEFA and TG than the control. The TG level was significantly lower for 6-week treatment (ASX: 304.8 ± 10.6; OL: 414.8 ± 9.5 mg/dl). Effect of ASX-O on lipid metabolic parameters, ex vivo After the treatment period, the non-fasting blood plasma in the ASX-O-treated SHRcp group showed a significant increase in
In this study, we investigated the effects of the oral administration of ASX-O in a MetS animal model of spontaneously hypertensive corpulent rat strain SHR/NDmrccp, which exhibits typical characteristics of this syndrome (Michaelis et al., 1984). The effects on the metabolic indices of MetS were monitored throughout a 22-week treatment period. In this study, ASX significantly lowered the blood pressure, similar to our previous findings (Hussein et al., 2005b,a), and showed improving effects on glucose and lipid metabolic parameters. One of the most crucial findings in this study was that ASX significantly lowered the blood glucose level of SHRcp in insulin tolerance tests, suggesting that ASX ameliorated InsR and improved insulin sensitivity. The effect on InsR was also significant, as shown by the HOMA-IR index, which is an
Fig. 7. Representative morphology of retroperitoneal white adipose tissue in control OL-treated SHRcp (I) and ASX-O-treated SHRcp (II). Fat samples taken from a similar position as periperitoneal fat deposit were fixed, sectioned, and hematoxylin-eosin stained.
G. Hussein et al. / Life Sciences 80 (2007) 522–529
indicator of InsR, and closely correlated with insulin sensitivity (Matthews et al., 1985; Pickavance et al., 1999). Our new finding is further supported by previous reports that ASX protects pancreatic β−cells against glucose toxicity by preventing the progressive destruction of these cells in diabetic db/db mice (Uchiyama et al., 2002). Another important finding is that ASX showed an ameliorative and improving effect on lipid metabolic indices in SHRcp. ASX significantly increased the HDL-cholesterol level in treated animals. It also significantly improved the levels of NEFA and TG, and the adiponectin marker. Lipid metabolic parameters have been widely described to play a crucial role in the development and progression of MetS and its consequent sequelae. Of these parameters, the adipocytokine hormone adiponectin has been shown to reduce blood glucose concentrations and increase fatty acids oxidation in the muscle (Fruebis et al., 2001) and to regulate (Hotamisligil et al., 1993) and ameliorate InsR (Yamauchi et al., 2003). HDL-cholesterol is one of the major determinant risk factors in MetS (Alberti et al., 2005; WHO, 1999), and its raised level in the blood was reported to retard the development of atherosclerosis, and cardiovascular sequelae in human epidemiological studies (Gordon et al., 1986) and in animal studies (Benoit et al., 1999; Brewer, 2004). An elevated level of circulating free fatty acids (FFA) is a characteristic feature of type 2 diabetes, and is strongly implicated in InsR development and β-cell dysfunction (Paolisso et al., 1995; Reaven et al., 1988). FFA were also reported as a risk marker for long-term development of glucose intolerance, the progression of type 2 diabetes (Charles et al., 1997), and as an important link for obesity, InsR, and type 2 diabetes (Boden, 1997). An elevated TG level is another major characteristic abnormality in diabetic dyslipidemia (Ginsberg et al., 2005), and in several studies, animals and humans, it has been reported to have a close correlation with InsR (Pan et al., 1997; Storlien et al., 1991). These parameters were progressively ameliorated by ASX-O administration. Moreover, the data showed that the HOMA-IR index is closely correlated to these metabolic changes of the lipid profiles. It is also worth mentioning that ASX-O ameliorated MetS features without affecting body weight. This can be explained, in part, by the large body of evidence that the adiponectin relationship with lipid metabolism is independent of body fat mass (Baratta et al., 2004; Maeda et al., 2002). From these results, it is unlikely that the combined effects of ASX-O on MetS include body weight reduction. Nevertheless, ASX-O showed a significant effect on adipose tissue by inhibiting the proliferation of white adipose tissue, as indicated by the decreased size of adipocytes. White adipocytes play a wide-ranging role in metabolic regulation and physiological homeostasis (Kershaw and Flier, 2004; Trayhurn and Beattie, 2001). Increased fat cell size is highly associated with InsR, and the development of diabetes, and may represent the failure of the adipose tissue mass to expand, and therefore to accommodate increased energy influx (Heilbronn et al., 2004). Evidence from human and animal studies substantiates the importance of adipose tissue, where too little, as in lipodystrophy (Seip and Trygstad, 1996), or too much, as in obesity (Brunzell and Hokanson, 1999), leads to InsR and diabetes.
527
The results showed a clear qualitative change reflected as an increase in cellularity as shown visually on the micrographs, where clusters of small adipocytes are clearly dispersed throughout the fat tissue. This adipogenic effect, which results in the generation of small fat cells, may explain the decrease in circulating FFA and the improvement in adiponectin and InsR. These cellular effects are most likely attributed to regulatory factors, mainly peroxisome proliferator-activated receptors gamma, which are widely reported to reduce circulating levels of FFA and stimulate adipocyte differentiation, thus favoring the formation of smaller, more insulin-sensitive adipocytes (Schoonjans et al., 1996; Spiegelman, 1998; Vazquez et al., 2002). This observed effect of ASX-O on white adipose tissue may add further support to ASX potential against MetS. On the other hand, ASX-O did not show significant effects on blood cell counts except for the WBC count, which was significantly decreased. In relation to MetS, such a decrease in WBC indicates an improving effect on the syndrome since increased WBC count has been reported to be associated with a variety of MetS features in clinical population studies (Lohsoonthorn et al., 2006; Wang et al., 2004). It may also be attributed to the consequent effects of ASX on the WBC, due to its antiinflammatory property (Ohgami et al., 2003). Considering the finding that ASX-O did not show a significant effect on insulin level, it is unlikely that ASX induces its effect on InsR directly on the blood insulin level. It is widely accepted that, although InsR leads to a decrease in glucose uptake, the relationship between glucose and insulin is quite complex and involves the interaction of many metabolic and regulatory factors (Radziuk, 2000). In this study, ASX exhibited significant ameliorative effects on MetS features in the dose of 50 mg/kg but not in the dose of 5 mg/kg (data not shown). Therefore, the dose–response relationship of ASX may need to be further investigated. In conclusion, our results indicate that ASX-O ameliorates InsR by mechanisms involving the increase of glucose uptake, and by modulating the levels of circulating adiponectin and blood lipids. These findings suggest ASX as a promising natural candidate in lifestyle disease protection and in health promotion. However, further studies on the molecular action mechanisms as well as clinical investigations are strongly recommended. Acknowledgements This work was supported by a grant from the Regional R and D Consortium Project sponsored by the Ministry of Economy, Trade and Industry, Japan. It was also supported, in part, by a grant from the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER). We thank Fuji Chemical Industry Co., Ltd., Toyama for supplying the astaxanthin product. The authors also acknowledge Ms Ayano Yanaga (Faculty of Medicine, University of Toyama) for her assistance in some of the experiments. References Alberti, K.G., Zimmet, P., Shaw, J., IDF Epidemiology Task Force Consensus Group, 2005. The metabolic syndrome—a new worldwide definition. Lancet 366 (9491), 1059–1062.
528
G. Hussein et al. / Life Sciences 80 (2007) 522–529
Baratta, R., Amato, S., Degano, C., Farina, M.G., Patane, G., Vigneri, R., Frittitta, L., 2004. Adiponectin relationship with lipid metabolism is independent of body fat mass: evidence from both cross-sectional and intervention studies. The Journal of Clinical Endocrinology and Metabolism 89 (6), 2665–2671. Benoit, P., Emmanuel, F., Caillaud, J.M., Bassinet, L., Castro, G., Gallix, P., Fruchart, J.C., Branellec, D., Denefle, P., Duverger, N., 1999. Somatic gene transfer of human ApoA-I inhibits atherosclerosis progression in mouse models. Circulation 99 (1), 105–110. Boden, G., 1997. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46 (1), 3–10. Brewer, H.B., 2004. High-density lipoproteins: a new potential therapeutic target for the prevention of cardiovascular disease. Arteriosclerosis, Thrombosis, and Vascular Biology 24 (3), 387–391. Brunzell, J.D., Hokanson, J.E., 1999. Dyslipidemia of central obesity and insulin resistance. Diabetes Care 22 (Suppl 3), C10–C13. Charles, M.A., Eschwege, E., Thibult, N., Claude, J.R., Warnet, J.M., Rosselin, G.E., Girard, J., Balkau, B., 1997. The role of non-esterified fatty acids in the deterioration of glucose tolerance in Caucasian subjects: results of the Paris Prospective Study. Diabetologia 40 (9), 1101–1106. Eckel, R.H., Grundy, S.M., Zimmet, P.Z., 2005. The metabolic syndrome. Lancet 365 (9468), 1415–1428. Fruebis, J., Tsao, T.S., Javorschi, S., Ebbets-Reed, D., Erickson, M.R., Yen, F.T, Bihain, B.E., Lodish, H.F., 2001. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proceedings of the National Academy of Sciences of the United States of America 98 (4), 2000–2010. Ginsberg, H.N., Zhang, Y.L., Hernandez-Ono, A., 2005. Regulation of plasma triglycerides in insulin resistance and diabetes. Archives of Medical Research 36 (3), 232–240. Gordon, D.J., Knoke, J., Probstfield, J.L., Superko, R., Tyroler, H.A., 1986. High-density lipoprotein cholesterol and coronary heart disease in hypercholesterolemic men: the Lipid Research Clinics Coronary Primary Prevention Trial. Circulation 74 (6), 1217–1225. Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends in Biotechnology 21 (5), 210–216. Heilbronn, L., Smith, S.R., Ravussin, E., 2004. Failure of fat cell proliferation, mitochondrial function and fat oxidation results in ectopic fat storage, insulin resistance and type II diabetes mellitus. International Journal of Obesity 28 (Suppl 4), S12–S21. Hotamisligil, G.S., Shargill, N.S., Spiegelman, B.M., 1993. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259 (5091), 87–91. Hussein, G., Goto, H., Oda, S., Iguchi, T., Sankawa, U., Matsumoto, K., Watanabe, H., 2005a. Antihypertensive potential and mechanism of action of astaxanthin: II. Vascular reactivity and hemorheology in spontaneously hypertensive rats. Biological & Pharmaceutical Bulletin 28 (6), 967–971. Hussein, G., Nakamura, M., Zhao, Q., Iguchi, T., Goto, H., Sankawa, U., Watanabe, H., 2005b. Antihypertensive and neuroprotective effects of astaxanthin in experimental animals. Biological & Pharmaceutical Bulletin 28 (1), 47–52. Hussein, G., Goto, H., Oda, S., Sankawa, U., Matsumoto, K., Watanabe, H., 2006. Antihypertensive potential and mechanism of action of astaxanthin: III. Antioxidant and histopathological effects in spontaneously hypertensive rats. Biological & Pharmaceutical Bulletin 29 (4), 684–688. Kershaw, E.E., Flier, J.S., 2004. Adipose tissue as an endocrine organ. The Journal of Clinical Endocrinology and Metabolism 89 (6), 2548–2556. Lohsoonthorn, V., Dhanamun, B., Williams, M.A., 2006. Prevalence of metabolic syndrome and its relationship to white blood cell count in a population of Thai men and women receiving routine health examinations. American Journal of Hypertension 19 (4), 339–345. Lorenz, R.T., Cysewski, G.R., 2000. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends in Biotechnology 18 (4), 160–167. Maeda, N., Shimomura, I., Kishida, K., Nishizawa, H., Matsuda, M., Nagaretani, H., Furuyama, N., Kondo, H., Takahashi, M., Arita, Y., Komuro, R., Ouchi, N., Kihara, S., Tochino, Y., Okutomi, K., Horie, M., Takeda, S., Aoyama, T.,
Funahashi, T., Matsuzawa, Y., 2002. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nature Medicine 8 (7), 731–737. Matthews, D.R., Hosker, J.P., Rudenski, A.S., Naylor, B.A., Treacher, .D.F., Turner, R.C., 1985. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28 (7), 412–419. Mertz, W., 1993. Chromium in human nutrition: a review. The Journal of Nutrition 123 (4), 626–633. Michaelis, O.E., Ellwood, K.C., Judge, J.M., Schoene, N.W., Hansen, C.T., 1984. Effect of dietary sucrose on the SHR/N-corpulent rat: a new model for insulin-independent diabetes. The American Journal of Clinical Nutrition 39 (4), 612–618. Moller, D., 2001. New drug targets for type 2 diabetes, and the metabolic syndrome. Nature 414 (6865), 821–827. Nakaya, Y., Minami, A., Harada, N., Sakamoto, S., Niwa, Y., Ohnaka, M., 2000. Taurine improves insulin sensitivity in the Otsuka Long–Evans Tokushima fatty rat, a model of spontaneous type 2 diabetes. The American Journal of Clinical Nutrition 71 (1), 54–58. Ohgami, K., Shiratori, K., Kotake, S., Nishida, T., Mizuki, N., Yazawa, K., Ohno, S., 2003. Effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo. Investigative Ophthalmology and Visual Science 44 (6), 2694–2701. Pan, D.A., Lillioja, S., Kriketos, A.D., Milner, M.R., Baur, L.A., Bogardus, C., Jenkins, A.B., Storlien, L.H., 1997. Skeletal muscle triglyceride levels are inversely related to insulin action. Diabetes 46 (6), 983–988. Paolisso, G., Tataranni, P.A., Foley, J.E., Bogardus, C., Howard, B.V., Ravussin, E., 1995. A high concentration of fasting plasma non-esterified fatty acids is a risk factor for the development of NIDDM. Diabetologia 38 (10), 1213–1217. Peth, J.A., Kinnick, T.R., Youngblood, E.B., Tritschler, H.J., Henriksen, E.J., 2000. Effects of a unique conjugate of alpha-lipoic acid and gamma-linolenic acid on insulin action in obese Zucker rats. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 278 (2), R453–R459. Pickavance, L.C., Tadayyon, M., Widdowson, P.S., Buckingham, R.E., Wilding, J.P., 1999. Therapeutic index for rosiglitazone in dietary obese rats: separation of efficacy and haemodilution. British Journal of Pharmacology 128 (7), 1570–1576. Radziuk, J., 2000. Insulin sensitivity and its measurement: structural commonalities among the methods. The Journal of Clinical Endocrinology and Metabolism 85 (12), 4426–4433. Reaven, G.M., Hollenbeck, C., Jeng, C.Y., Wu, M.S., Chen, Y.D., 1988. Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37 (8), 1020–1024. Schoonjans, K., Staels, B., Auwerx, J., 1996. The peroxisome proliferators activated receptors (PPARs) and their effects on lipid metabolism and adipocytes differentiation. Biochimica et Biophysica Acta 1302 (2), 93–109. Scott, C., 2003. Diagnosis, prevention, and intervention for the metabolic syndrome. The American Journal of Cardiology 92 (1A), 35i–42i. Seip, M., Trygstad, O., 1996. Generalized lipodystrophy, congenital and acquired lipoatrophy. Acta Paediatrica, Supplementum 413, 2–28. Shepherd, J., Betteridge, J., Van Gaal, L., Panel, E.C., 2005. Nicotinic acid in the management of dyslipidaemia associated with diabetes and metabolic syndrome: a position paper developed by a European Consensus Panel. Current Medical Research and Opinion 21 (5), 665–682. Spiegelman, B.M., 1998. PPAR-gamma: adipogenic regulator and thiazolidinediones receptor. Diabetes 47 (4), 507–514. Storlien, L.H., Jenkins, A.B., Chisholm, D.J., Pascoe, W.S., Khouri, S., Kraegen, E.W., 1991. Influence of dietary fat composition on development of insulin resistance in rats: relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipids. Diabetes 40 (2), 280–289. Trayhurn, P., Beattie, J.H., 2001. Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. The Proceedings of the Nutrition Society 60 (3), 329–339. Uchiyama, K., Naito, Y., Hasegawa, G., Nakamura, N., Takahashi, J., Yoshikawa, T., 2002. Astaxanthin protects beta-cells against glucose toxicity in diabetic db/db mice. Redox Report: Communications in Free Radical Research 7 (5), 290–293. Vazquez, M., Silvestre, J., Prous, J., 2002. Experimental approaches to study PPAR gamma agonists and antidiabetic drugs. Methods and Findings in Experimental and Clinical Pharmacology 48 (8), 515–523.
G. Hussein et al. / Life Sciences 80 (2007) 522–529 Wang, Y., Lin, S., Liu, P., Cheung, B., Lai, W., 2004. Association between hematological parameters and metabolic syndrome components in a Chinese population. Journal of Diabetes and its Complications 18 (6), 322–327. WHO, 1999. Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications: Report of a WHO Consultation, Geneva, Switzerland: Department of Noncommunicable Disease Surveillance. World Health Organization. Xu, M., Xiao, S., Sun, Y., Zheng, X., Ou-Yang, Y., Guan, C., 2005. The study of anti-metabolic syndrome effect of puerarin in vitro. Life Sciences 77 (25), 3183–3196.
529
Yamauchi, T., Kamon, J., Waki, H., Imai, Y., Shimozawa, N., Hioki, K., Uchida, S., Ito, Y., Takakuwa, K., Matsui, J., Takata, M., Eto, K., Terauchi, Y., Komeda, K., Tsunoda, M.,Murakami,K., Ohnishi,Y., Naitoh, T., Yamamura, K., Ueyama, Y., Froguel, P., Kimura, S., Nagai, R., Kadowaki, T., 2003. Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis. The Journal of Biological Chemistry 278, 2461–2468.
Astaxanthin ameliorates features of metabolic syndrome in SHR/NDmcr-cp Ghazi Hussein a,b,⁎, Takako Nakagawa c , Hirozo Goto d , Yutaka Shimada d , Kinzo Matsumoto b , Ushio Sankawa a , Hiroshi Watanabe a a
d
International Research Center for Traditional Medicine, Toyama, Toyama Prefecture 939-8224, Japan b Division of Medicinal Pharmacology, Institute of Natural Medicine, Japan c Department of Kampo Diagnostics, Institute of Natural Medicine, Japan Department of Japanese Oriental Medicine, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan Received 29 May 2006; accepted 28 September 2006
Abstract Glucose and lipid metabolic parameters play crucial roles in metabolic syndrome and its major feature of insulin resistance. This study was designed to investigate whether dietary astaxanthin oil (ASX-O) has potential effects on metabolic syndrome features in an SHR/NDmcr-cp (cp/ cp) rat model. Oral administration of ASX (50 mg/kg/day) for 22 weeks induced a significant reduction in arterial blood pressure in SHRcp. It also significantly reduced the fasting blood glucose level, homeostasis index of insulin resistance (HOMA-IR), and improved insulin sensitivity. The results also showed an improved adiponectin level, a significant increase in high-density lipoprotein cholesterol, a significant decrease in plasma levels of triglycerides, and non-esterified fatty acids. Additionally, ASX showed significant effects on the white adipose tissue by decreasing the size of the fat cells. These results suggest that ASX ameliorates insulin resistance by mechanisms involving the increase of glucose uptake, and by modulating the level of circulating lipid metabolites and adiponectin. © 2006 Elsevier Inc. All rights reserved. Keywords: Astaxanthin; Metabolic syndrome; Blood pressure; Insulin resistance; Lipids; Adiponectin; SHR/NDmcr-cp
Introduction Metabolic syndrome (MetS) is a worldwide-defined (Alberti et al., 2005; WHO, 1999) lifestyle disease that is described as a clustering of multiple metabolic abnormalities, and cardiovascular risk factors including hypertension, obesity, hyperlipidemia, and insulin resistance (InsR) (Eckel et al., 2005). InsR is considered as the central component of this cluster, and is described as a condition whereby there is resistance to insulinmediated glucose uptake by cells. Glucose toxicity (chronic hyperglycemia), and chronically elevated levels of free fatty acids are major metabolic features of MetS. Recently, MetS has become a focus of a number of scientific studies aiming at its prevention, and intervention (Moller, 2001). Current conven⁎ Corresponding author. Division of Medicinal Pharmacology, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Tel.: +81 76 434 7611; fax: +81 76 434 5056. E-mail address: [email protected] (G. Hussein). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.09.041
tional treatment of MetS may include one or more pharmacological therapies, mainly statins (3-hydroxy-3-methylglutaryl coenzyme A inhibitors), angiotensin-converting enzyme inhibitors, fibrates, and thiazolidinediones (Scott, 2003). Diet and lifestyle changes are considered crucial factors in the prevention and treatment. So far, very few reports have described natural products and medicines as candidates against MetS (Shepherd et al., 2005; Xu et al., 2005) and InsR (Mertz, 1993; Nakaya et al., 2000; Peth et al., 2000). Astaxanthin (ASX) is a powerful antioxidant, xanthophyll carotenoid, commonly found in seafood, and predominantly produced by microalga Haematococcus pluvialis Flotow (Lorenz and Cysewski, 2000), which can accumulate more than 30 g of ASX per kg dry biomass (Guerin et al., 2003). We have reported the antihypertensive effects (Hussein et al., 2005b,a, 2006) and action mechanisms (Hussein et al., 2005a) of a dietary carotenoid astaxanthin (ASX-O) in SHR. In this study, we investigated the effects of ASX-O on MetS in SHR/NDmcr-cp (cp/cp), a spontaneously hypertensive rat model of MetS (referred to as
G. Hussein et al. / Life Sciences 80 (2007) 522–529
523
Table 2 The effect of ASX-O administration on body weight in treated animals Treatment period (week)
0 3 6 9 12 15 18 21
Body weight (g) Wistar-OL
SHRcp-OL
SHRcp-ASX
153 ± 1 245 ± 2 317 ± 2 356 ± 3 387 ± 3 402 ± 3 422 ± 3 411 ± 3
149 ± 3 243 ± 7 335 ± 1 405 ± 2 465 ± 3 503 ± 3 539 ± 4 546 ± 3
150 ± 4 244 ± 2 337 ± 2 406 ± 2 463 ± 3 501 ± 2 531 ± 3 533 ± 3
Each value represents the mean ± S.E.M. (n = 6). Fig. 1. Effects of oral administration of ASX-O on systolic blood pressure in SHRcp. The animals received a control vehicle [OL (1 ml/kg/day), open circles], or ASX-O [ASX (50 mg/kg/day), closed circles] for an 18-week treatment period. Each data point represents the mean ± S.E.M. of 6 rats per group. ⁎p b 0.05; ⁎⁎p b 0.01 vs. the control group (t-test).
All the reagents used in these experiments were of analytical grade as follows: Pentobarbital sodium (Tokyo Chemical Industry, Tokyo, Japan), insulin (Novolin R 100, Novo Nordisk, Tokyo), heparin (Novo-Heparin, Mochida, Tokyo), and sodium citrate (Fuso, Osaka, Japan).
on: 07:30−19:30). The animals were housed for at least 1 week before the experiments, and fed a normal diet (Lab MR, NOSAN, Yokohama, Japan) and water ad libitum. Body weight and food intake (FI) were measured daily during the experimental period. ASX-O, composed of 4.5–5.5% (weight per weight) of ASX in an edible oil base, was obtained from Fuji Chemical (Fuji Chemical Industry Co., Ltd, Toyama, Japan), dissolved, and diluted in olive oil (OL) (Wako Pure Chemicals, Osaka, Japan). Administered doses were calculated on the basis of the amount of ASX in the batches of dietary ASX-O. The animals were divided into three groups, a normal Wistar group and two SHRcp groups (6 rats/ group), and were treated once daily for 22 weeks. The ASX-Otreated SHRcp group was administered daily ASX [50 mg (83.8 mmol)/kg, intubation by p.o.], which was measured as 0.1 ml of the stock/100 g bodyweight and diluted, for convenience and acceptability, by an equivalent volume of olive oil prior to administration. The other SHRcp-group and the Wistar group were adopted as control groups and were similarly treated with OL (1 ml/kg/day). All experimental procedures were performed in accordance with the standards established by the ‘Guide for the Care and Use of Laboratory Animals at University of Toyama’.
Animals and drug treatment
Measurement of blood pressure, and heart rate
Male SHR/NDmrc-cp (cp/cp) (designated as SHRcp), and standard Wistar (Wistar/ST) (6 weeks old) rats were used in this study. The animals were obtained from colonies of specific pathogen-free rats maintained by Japan SLC (Shizuoka, Japan). Housing conditions were thermostatically maintained at 24 ± 1 °C with constant humidity (60%) and a 12 h-light/dark cycle (light
Arterial blood pressure and heart rate were determined by a tail cuff system, as previously reported (Hussein et al., 2005b). Briefly, conscious rats were lightly supported in a holder made of cloth mesh and maintained at 37 ± 1 °C (Model THC-1 Digital Thermo, Softron, Tokyo, Japan). Blood pressure from the tail artery was indirectly measured using tail-cuff apparatus (BP-98, Softron), which was controlled with a personal computer. Values are presented as the average of three independent measurements.
SHRcp). A 22-week administration period of ASX at doses of 5 and 50 mg/kg/day, p.o., was examined in SHRcp. In this study, significant changes in the features of MetS were produced by the dose of 50 mg/kg. The effects of ASX-O on blood pressure, blood glucose, blood insulin, insulin sensitivity, and blood lipids were investigated. To the best of our knowledge, this is the first study on the effects of ASX, and carotenoids in general, on MetS. Materials and methods Chemicals
Table 1 Effects of ASX-O on blood pressure and heart rate in SHRcp after treatment for 18 weeks
Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean blood pressure (mmHg) Heart rate (beat/min)
Control (OL)
ASX-O
208 ± 3 179 ± 3 188 ± 3 396 ± 25
189 ± 5⁎ 156 ± 5⁎⁎ 167 ± 1⁎⁎ 374 ± 29
Each value represents the mean ± S.E.M. (n = 6). ⁎p b 0.05; ⁎⁎p b 0.01 vs. the control group (t-test).
Measurement of blood cell counts Blood from the heart of each sacrificed SHRcp was separately collected into heparinized syringes containing 5% heparin, and 2% sodium citrate, and was instantly and gently mixed. A small portion of the blood was used for the cell count, which was determined by a cell counter (Celltac α, Nihon Kohden, Tokyo). The other portion of blood was immediately centrifuged at 3000 g
524
G. Hussein et al. / Life Sciences 80 (2007) 522–529
Table 3 Blood cell counts in SHRcp after treatment with ASX-O for 22 weeks Group
WBC (×102/μl)
RBC (×104/μl)
HGB (g/dl)
HCT (%)
MCV (f l)
MCH (pg)
MCHC (g/dl)
PLT (×104/μl)
Control ASX-O
59.3 ± 1.5 41.0 ± 2.3⁎
950.0 ± 3.1 943.8 ± 4.2
14.9 ± 0.3 15.0 ± 0.2
46.9 ± 0.9 47.4 ± 0.5
49.2 ± 0.6 49.5 ± 0.5
15.6 ± 0.2 15.7 ± 0.2
31.7 ± 0.1 31.7 ± 0.2
72.2 ± 5.3 72.5 ± 0.9
Blood cell count was performed in SHR treated with the control OL (1 ml/kg/day) and ASX (50 mg/kg/day) for 22 weeks. WBC, white blood cell count; RBC, red BC; HGB, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, MC hemoglobin; MCHC, MCH concentration; PLT, platelet count. Each value represents the mean ± S.E.M. (n = 6). ⁎p b 0.01 vs. the control group (t-test).
for 15 min (Kubota 8700, Kubota, Tokyo), and the plasma was separated and used for analysis of the metabolic parameters. Measurement of blood glucose level All rats were fasted overnight with free access to water, and then anesthetized by ethyl ether inhalation. Blood glucose measurement was carried out at 0, 6, 12, and 18 weeks of treatment. Blood was withdrawn from the tail, and instantly tested using a blood glucose-measuring device Antisense III (Horiba, Tokyo, Japan).
Blood sample assays Blood insulin and adiponectin levels were measured using enzyme-linked immunosorbent assay kits (Morinaga Institute of Biological Science, Yokohama, Japan; and Otsuka Pharmaceuticals, Tokyo, Japan, respectively). Blood high-density lipoprotein (HDL)-cholesterol, free fatty acids (FFA), and triglyceride (TG) levels were determined by the HDL-cholesterol E, nonesterified fatty acid (NEFA) C-test, and TG L-type test (Wako, Osaka, Japan), respectively. Histological study
Insulin tolerance test
This model, which incorporates measures of both fasting plasma concentrations of glucose and insulin, was used to calculate the index of InsR as [insulin (μU/ml) × glucose (mM)/ 22.5] (Matthews et al., 1985).
After sacrifice of the animals, small pieces of retroperitoneal white adipose tissue were removed immediately, rinsed with saline and fixed with formalin (10%). Specimens from each rat were embedded in paraffin and sections were stained with hematoxylin and eosin. The specimens and slides were prepared by the Special Reference Laboratories (SRL Tokyo Medical Inc., Tokyo). To examine the size of white adipocytes, the number of adipocytes was counted in limited areas (capture size, 640X480 pixels, equivalent to 12,288 μm2) of each stained specimen. The mean value of the adipocyte count was designated as an index of the cell size; as a larger number means a smaller size. In this study, multilocular cells were excluded and fused cells were counted as a one cell. Examination and photography of the slides was carried out using a light microscopy system (Olympus Provis AX80,
Fig. 2. Fasting blood glucose level in the ASX-treated SHRcp group (closed circles), and the control OL-treated group: SHRcp (open circles), and Wistar (open triangles) rats. Data represent the mean ± S.E.M. (n = 5–6). ⁎p b 0.001 vs. the SHRcp control (t-test).
Fig. 3. Blood glucose level in the ASX-treated SHRcp group (closed circles), and the control OL-treated group: SHRcp (open circles) and Wistar (open triangles) rats, at the insulin tolerance test. Data are the mean ± S.E.M. (n = 5–6). ⁎p b 0.05 vs. the control (t-test).
Tested rats were fasted as above and then injected with insulin 1 ml/kg body weight (equivalent to 0.5 U/kg), i.p. After the injection, blood was obtained from the tail into separate heparinized syringes at intervals of 0 min, 30, 60, and 120 min. Each withdrawn blood sample was immediately tested for blood glucose, as mentioned above. Homeostasis model assessment of insulin resistance (HOMA-IR)
G. Hussein et al. / Life Sciences 80 (2007) 522–529
525
Table 4 Time-course of the effects of ASX-O on glucose metabolic parameters, measured from fasting blood plasma of SHRcp Metabolic parameters
Treatment (week)
Wistar-OL
SHRcp-OL
SHRcp-ASX
Blood glucose (mg/dl)
0 6 18 0 6 18 0 6 18 0 6 18
137.0 ± 15.1 95.3 ± 2.4 128.5 ± 2.3 0.04 ± 0.00 0.09 ± 0.02 0.19 ± 0.03 0.28 ± 0.04 0.52 ± 0.10 1.18 ± 0.09 4.81 ± 0.28 4.44 ± 0.59 4.87 ± 0.29
138.3 ± 4.8 91.7 ± 4.3 154.7 ± 0.9 0.03 ± 0.01 1.47 ± 0.07 1.78 ± 0.04 0.23 ± 0.09 7.88 ± 0.15 16.15 ± 0.30 7.38 ± 0.27 5.91 ± 0.16 5.18 ± 0.15
135.0 ± 2.1 89.3 ± 8.2 123.7 ± 0.3⁎ 0.28 ± 0.03 1.61 ± 0.06 1.63 ± 0.07 2.23 ± 0.15 8.39 ± 0.50 11.87 ± 0.50⁎ 7.08 ± 0.43 8.21 ± 0.14⁎ 5.84 ± 0.29
Blood insulin (ng/ml) HOMA-IR
Adiponectin (μg/ml)
HOMA-IR, homeostasis model assessment of insulin resistance. Each value represents the mean ± S.E.M. (n = 4). ⁎p b 0.01 vs. the SHRcp control (t-test).
Olympus Optical Co. Ltd., Tokyo). Calibration was carried out using a standard area of (1/20 × 1/20 mm) (Burker–Turk haemocytometer, JIS No. E3871, Kayagaki Irika Kogyo Co., LTD., Tokyo). Histological images were measured and analyzed blindly by two investigators using the UTHSCSA Image Tool for Windows, version 3.00 (San Antonio, Texas, USA). Statistical analysis Statistical significance was determined by Student's t-test or Mann–Whitney Rank Sum Test for unpaired observations. Oneway analysis of variance (ANOVA) was performed for multiple comparisons between groups. Differences with p b 0.05 were considered statistically significant. Results Effects of ASX-O on blood pressure In this study, ASX-O showed a significant blood pressurelowering effect on arterial blood pressure, as represented by the effect on systolic blood pressure depicted in Fig. 1 and summarized in Table 1. Throughout the treatment period, the daily administered ASX-O showed no significant or consistent effect on the heart rate. The body weight of ASX-O-treated SHRcp did not significantly change, compared to the SHRcp-OL control group (Table 2). However, the SHRcp groups showed significantly higher body weight than the normal Wistar group, starting from 11 weeks of age ( p b 0.01), and exceeding a 100 g difference from 22 weeks of age ( p b 0.001) (at the age of 25 weeks: Wistar, 420 ± 4 g; SHRcp, 534 ± 4 g). Similarly, no significant differences in daily FI were found among SHRcp groups, which also showed a higher FI than the Wistar group throughout the study (at 25 weeks: Wistar, 19 ± 0.4 g; SHRcp, 21 ± 0.7 g).
Fig. 4. High-density lipoprotein (HDL)-cholesterol level in ASX-O- and control-treated SHRcp, ex vivo. Data are the mean ± S.E.M. (n = 4−5). ⁎p b 0.01 vs. the control (t-test).
except for the white blood cell (WBC) count, which was significantly decreased (Table 3). Effect of ASX-O on blood glucose level The ASX group showed a lower blood glucose level than the corresponding OL group (123.7 ± 0.3 and 154.7 ± 0.9 mg/dl, respectively). The effect was significant and comparable to the normal level in the Wistar group in the 18th week of treatment (Fig. 2). Effect of ASX-O on InsR using insulin tolerance test In insulin tolerance tests, 60 min after the insulin injection, the blood glucose level significantly dropped in the ASX group compared to the OL group. This effect was also maintained at 120 min (Fig. 3), indicating improved insulin sensitivity in SHRcp in the ASX-treated group compared to the control group.
Blood cell counts The 22-week treatment of SHRcp with ASX (50 mg/kg) did not change the blood cell count indices compared to the control,
Fig. 5. Non-esterified fatty acids (NEFA) level in ASX-O- and control-treated animal groups, ex vivo. Data represent the mean ± S.E.M. (n = 4−5). ⁎p b 0.001 vs. the control (t-test).
526
G. Hussein et al. / Life Sciences 80 (2007) 522–529
HDL-cholesterol level, compared to the control (77.77 ± 2.52 and 63.87 ± 2.32 mg/dl, respectively) (Fig. 4). It also exhibited a significantly lower level of NEFA compared to the corresponding control (0.71 ± 0.04 and 1.07 ± 0.07 mEq/l, respectively) (Fig. 5). The ASX-O-treated group also exhibited a significant decrease in TG level compared to the OL group of SHRcp (360.8 ± 24.2 and 467.1 ± 11.8 mg/dl, respectively) (Fig. 6). Effect of ASX-O on adipose tissue
Fig. 6. Triglyceride (TG) level in ASX-O- and control-treated animals, ex vivo. Data represent the mean ± S.E.M. (n = 4). ⁎p b 0.01 vs. the control (t-test).
Time-course of the effects of ASX-O on glucose metabolic parameters
In microscopic studies (Fig. 7), white adipose tissue of the ASX-treated group showed large numbers and aggregates of small fat cells, clearly distinguished as discrete clusters or patches of cells. After treatment with ASX-O for 22 weeks in SHRcp, the number of fat cells was significantly higher in the ASX group than the control [SHRcp-ASX group: 184 ± 3; SHRcp-OL: 140 ± 3 cells/12288 μm2 (mean ± S.E.M.), n = 5–6, p b 0.01, t-test]. Discussion
During a 0–18 week-treatment period, fasting blood plasma of SHRcp showed lower levels of blood glucose and blood insulin in the ASX group compared to the control OL group. The effect was significant on blood glucose after 18-week administration (Table 4). Plasma adiponectin level was higher in the ASX group than that in the control group, with a significant level in the 6th week (8.2 ± 0.1 and 5.9 ± 0.2 g/ml, respectively). The ASX-O-treated group exhibited lower plasma levels of NEFA and TG than the control. The TG level was significantly lower for 6-week treatment (ASX: 304.8 ± 10.6; OL: 414.8 ± 9.5 mg/dl). Effect of ASX-O on lipid metabolic parameters, ex vivo After the treatment period, the non-fasting blood plasma in the ASX-O-treated SHRcp group showed a significant increase in
In this study, we investigated the effects of the oral administration of ASX-O in a MetS animal model of spontaneously hypertensive corpulent rat strain SHR/NDmrccp, which exhibits typical characteristics of this syndrome (Michaelis et al., 1984). The effects on the metabolic indices of MetS were monitored throughout a 22-week treatment period. In this study, ASX significantly lowered the blood pressure, similar to our previous findings (Hussein et al., 2005b,a), and showed improving effects on glucose and lipid metabolic parameters. One of the most crucial findings in this study was that ASX significantly lowered the blood glucose level of SHRcp in insulin tolerance tests, suggesting that ASX ameliorated InsR and improved insulin sensitivity. The effect on InsR was also significant, as shown by the HOMA-IR index, which is an
Fig. 7. Representative morphology of retroperitoneal white adipose tissue in control OL-treated SHRcp (I) and ASX-O-treated SHRcp (II). Fat samples taken from a similar position as periperitoneal fat deposit were fixed, sectioned, and hematoxylin-eosin stained.
G. Hussein et al. / Life Sciences 80 (2007) 522–529
indicator of InsR, and closely correlated with insulin sensitivity (Matthews et al., 1985; Pickavance et al., 1999). Our new finding is further supported by previous reports that ASX protects pancreatic β−cells against glucose toxicity by preventing the progressive destruction of these cells in diabetic db/db mice (Uchiyama et al., 2002). Another important finding is that ASX showed an ameliorative and improving effect on lipid metabolic indices in SHRcp. ASX significantly increased the HDL-cholesterol level in treated animals. It also significantly improved the levels of NEFA and TG, and the adiponectin marker. Lipid metabolic parameters have been widely described to play a crucial role in the development and progression of MetS and its consequent sequelae. Of these parameters, the adipocytokine hormone adiponectin has been shown to reduce blood glucose concentrations and increase fatty acids oxidation in the muscle (Fruebis et al., 2001) and to regulate (Hotamisligil et al., 1993) and ameliorate InsR (Yamauchi et al., 2003). HDL-cholesterol is one of the major determinant risk factors in MetS (Alberti et al., 2005; WHO, 1999), and its raised level in the blood was reported to retard the development of atherosclerosis, and cardiovascular sequelae in human epidemiological studies (Gordon et al., 1986) and in animal studies (Benoit et al., 1999; Brewer, 2004). An elevated level of circulating free fatty acids (FFA) is a characteristic feature of type 2 diabetes, and is strongly implicated in InsR development and β-cell dysfunction (Paolisso et al., 1995; Reaven et al., 1988). FFA were also reported as a risk marker for long-term development of glucose intolerance, the progression of type 2 diabetes (Charles et al., 1997), and as an important link for obesity, InsR, and type 2 diabetes (Boden, 1997). An elevated TG level is another major characteristic abnormality in diabetic dyslipidemia (Ginsberg et al., 2005), and in several studies, animals and humans, it has been reported to have a close correlation with InsR (Pan et al., 1997; Storlien et al., 1991). These parameters were progressively ameliorated by ASX-O administration. Moreover, the data showed that the HOMA-IR index is closely correlated to these metabolic changes of the lipid profiles. It is also worth mentioning that ASX-O ameliorated MetS features without affecting body weight. This can be explained, in part, by the large body of evidence that the adiponectin relationship with lipid metabolism is independent of body fat mass (Baratta et al., 2004; Maeda et al., 2002). From these results, it is unlikely that the combined effects of ASX-O on MetS include body weight reduction. Nevertheless, ASX-O showed a significant effect on adipose tissue by inhibiting the proliferation of white adipose tissue, as indicated by the decreased size of adipocytes. White adipocytes play a wide-ranging role in metabolic regulation and physiological homeostasis (Kershaw and Flier, 2004; Trayhurn and Beattie, 2001). Increased fat cell size is highly associated with InsR, and the development of diabetes, and may represent the failure of the adipose tissue mass to expand, and therefore to accommodate increased energy influx (Heilbronn et al., 2004). Evidence from human and animal studies substantiates the importance of adipose tissue, where too little, as in lipodystrophy (Seip and Trygstad, 1996), or too much, as in obesity (Brunzell and Hokanson, 1999), leads to InsR and diabetes.
527
The results showed a clear qualitative change reflected as an increase in cellularity as shown visually on the micrographs, where clusters of small adipocytes are clearly dispersed throughout the fat tissue. This adipogenic effect, which results in the generation of small fat cells, may explain the decrease in circulating FFA and the improvement in adiponectin and InsR. These cellular effects are most likely attributed to regulatory factors, mainly peroxisome proliferator-activated receptors gamma, which are widely reported to reduce circulating levels of FFA and stimulate adipocyte differentiation, thus favoring the formation of smaller, more insulin-sensitive adipocytes (Schoonjans et al., 1996; Spiegelman, 1998; Vazquez et al., 2002). This observed effect of ASX-O on white adipose tissue may add further support to ASX potential against MetS. On the other hand, ASX-O did not show significant effects on blood cell counts except for the WBC count, which was significantly decreased. In relation to MetS, such a decrease in WBC indicates an improving effect on the syndrome since increased WBC count has been reported to be associated with a variety of MetS features in clinical population studies (Lohsoonthorn et al., 2006; Wang et al., 2004). It may also be attributed to the consequent effects of ASX on the WBC, due to its antiinflammatory property (Ohgami et al., 2003). Considering the finding that ASX-O did not show a significant effect on insulin level, it is unlikely that ASX induces its effect on InsR directly on the blood insulin level. It is widely accepted that, although InsR leads to a decrease in glucose uptake, the relationship between glucose and insulin is quite complex and involves the interaction of many metabolic and regulatory factors (Radziuk, 2000). In this study, ASX exhibited significant ameliorative effects on MetS features in the dose of 50 mg/kg but not in the dose of 5 mg/kg (data not shown). Therefore, the dose–response relationship of ASX may need to be further investigated. In conclusion, our results indicate that ASX-O ameliorates InsR by mechanisms involving the increase of glucose uptake, and by modulating the levels of circulating adiponectin and blood lipids. These findings suggest ASX as a promising natural candidate in lifestyle disease protection and in health promotion. However, further studies on the molecular action mechanisms as well as clinical investigations are strongly recommended. Acknowledgements This work was supported by a grant from the Regional R and D Consortium Project sponsored by the Ministry of Economy, Trade and Industry, Japan. It was also supported, in part, by a grant from the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER). We thank Fuji Chemical Industry Co., Ltd., Toyama for supplying the astaxanthin product. The authors also acknowledge Ms Ayano Yanaga (Faculty of Medicine, University of Toyama) for her assistance in some of the experiments. References Alberti, K.G., Zimmet, P., Shaw, J., IDF Epidemiology Task Force Consensus Group, 2005. The metabolic syndrome—a new worldwide definition. Lancet 366 (9491), 1059–1062.
528
G. Hussein et al. / Life Sciences 80 (2007) 522–529
Baratta, R., Amato, S., Degano, C., Farina, M.G., Patane, G., Vigneri, R., Frittitta, L., 2004. Adiponectin relationship with lipid metabolism is independent of body fat mass: evidence from both cross-sectional and intervention studies. The Journal of Clinical Endocrinology and Metabolism 89 (6), 2665–2671. Benoit, P., Emmanuel, F., Caillaud, J.M., Bassinet, L., Castro, G., Gallix, P., Fruchart, J.C., Branellec, D., Denefle, P., Duverger, N., 1999. Somatic gene transfer of human ApoA-I inhibits atherosclerosis progression in mouse models. Circulation 99 (1), 105–110. Boden, G., 1997. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46 (1), 3–10. Brewer, H.B., 2004. High-density lipoproteins: a new potential therapeutic target for the prevention of cardiovascular disease. Arteriosclerosis, Thrombosis, and Vascular Biology 24 (3), 387–391. Brunzell, J.D., Hokanson, J.E., 1999. Dyslipidemia of central obesity and insulin resistance. Diabetes Care 22 (Suppl 3), C10–C13. Charles, M.A., Eschwege, E., Thibult, N., Claude, J.R., Warnet, J.M., Rosselin, G.E., Girard, J., Balkau, B., 1997. The role of non-esterified fatty acids in the deterioration of glucose tolerance in Caucasian subjects: results of the Paris Prospective Study. Diabetologia 40 (9), 1101–1106. Eckel, R.H., Grundy, S.M., Zimmet, P.Z., 2005. The metabolic syndrome. Lancet 365 (9468), 1415–1428. Fruebis, J., Tsao, T.S., Javorschi, S., Ebbets-Reed, D., Erickson, M.R., Yen, F.T, Bihain, B.E., Lodish, H.F., 2001. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proceedings of the National Academy of Sciences of the United States of America 98 (4), 2000–2010. Ginsberg, H.N., Zhang, Y.L., Hernandez-Ono, A., 2005. Regulation of plasma triglycerides in insulin resistance and diabetes. Archives of Medical Research 36 (3), 232–240. Gordon, D.J., Knoke, J., Probstfield, J.L., Superko, R., Tyroler, H.A., 1986. High-density lipoprotein cholesterol and coronary heart disease in hypercholesterolemic men: the Lipid Research Clinics Coronary Primary Prevention Trial. Circulation 74 (6), 1217–1225. Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends in Biotechnology 21 (5), 210–216. Heilbronn, L., Smith, S.R., Ravussin, E., 2004. Failure of fat cell proliferation, mitochondrial function and fat oxidation results in ectopic fat storage, insulin resistance and type II diabetes mellitus. International Journal of Obesity 28 (Suppl 4), S12–S21. Hotamisligil, G.S., Shargill, N.S., Spiegelman, B.M., 1993. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259 (5091), 87–91. Hussein, G., Goto, H., Oda, S., Iguchi, T., Sankawa, U., Matsumoto, K., Watanabe, H., 2005a. Antihypertensive potential and mechanism of action of astaxanthin: II. Vascular reactivity and hemorheology in spontaneously hypertensive rats. Biological & Pharmaceutical Bulletin 28 (6), 967–971. Hussein, G., Nakamura, M., Zhao, Q., Iguchi, T., Goto, H., Sankawa, U., Watanabe, H., 2005b. Antihypertensive and neuroprotective effects of astaxanthin in experimental animals. Biological & Pharmaceutical Bulletin 28 (1), 47–52. Hussein, G., Goto, H., Oda, S., Sankawa, U., Matsumoto, K., Watanabe, H., 2006. Antihypertensive potential and mechanism of action of astaxanthin: III. Antioxidant and histopathological effects in spontaneously hypertensive rats. Biological & Pharmaceutical Bulletin 29 (4), 684–688. Kershaw, E.E., Flier, J.S., 2004. Adipose tissue as an endocrine organ. The Journal of Clinical Endocrinology and Metabolism 89 (6), 2548–2556. Lohsoonthorn, V., Dhanamun, B., Williams, M.A., 2006. Prevalence of metabolic syndrome and its relationship to white blood cell count in a population of Thai men and women receiving routine health examinations. American Journal of Hypertension 19 (4), 339–345. Lorenz, R.T., Cysewski, G.R., 2000. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends in Biotechnology 18 (4), 160–167. Maeda, N., Shimomura, I., Kishida, K., Nishizawa, H., Matsuda, M., Nagaretani, H., Furuyama, N., Kondo, H., Takahashi, M., Arita, Y., Komuro, R., Ouchi, N., Kihara, S., Tochino, Y., Okutomi, K., Horie, M., Takeda, S., Aoyama, T.,
Funahashi, T., Matsuzawa, Y., 2002. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nature Medicine 8 (7), 731–737. Matthews, D.R., Hosker, J.P., Rudenski, A.S., Naylor, B.A., Treacher, .D.F., Turner, R.C., 1985. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28 (7), 412–419. Mertz, W., 1993. Chromium in human nutrition: a review. The Journal of Nutrition 123 (4), 626–633. Michaelis, O.E., Ellwood, K.C., Judge, J.M., Schoene, N.W., Hansen, C.T., 1984. Effect of dietary sucrose on the SHR/N-corpulent rat: a new model for insulin-independent diabetes. The American Journal of Clinical Nutrition 39 (4), 612–618. Moller, D., 2001. New drug targets for type 2 diabetes, and the metabolic syndrome. Nature 414 (6865), 821–827. Nakaya, Y., Minami, A., Harada, N., Sakamoto, S., Niwa, Y., Ohnaka, M., 2000. Taurine improves insulin sensitivity in the Otsuka Long–Evans Tokushima fatty rat, a model of spontaneous type 2 diabetes. The American Journal of Clinical Nutrition 71 (1), 54–58. Ohgami, K., Shiratori, K., Kotake, S., Nishida, T., Mizuki, N., Yazawa, K., Ohno, S., 2003. Effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo. Investigative Ophthalmology and Visual Science 44 (6), 2694–2701. Pan, D.A., Lillioja, S., Kriketos, A.D., Milner, M.R., Baur, L.A., Bogardus, C., Jenkins, A.B., Storlien, L.H., 1997. Skeletal muscle triglyceride levels are inversely related to insulin action. Diabetes 46 (6), 983–988. Paolisso, G., Tataranni, P.A., Foley, J.E., Bogardus, C., Howard, B.V., Ravussin, E., 1995. A high concentration of fasting plasma non-esterified fatty acids is a risk factor for the development of NIDDM. Diabetologia 38 (10), 1213–1217. Peth, J.A., Kinnick, T.R., Youngblood, E.B., Tritschler, H.J., Henriksen, E.J., 2000. Effects of a unique conjugate of alpha-lipoic acid and gamma-linolenic acid on insulin action in obese Zucker rats. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 278 (2), R453–R459. Pickavance, L.C., Tadayyon, M., Widdowson, P.S., Buckingham, R.E., Wilding, J.P., 1999. Therapeutic index for rosiglitazone in dietary obese rats: separation of efficacy and haemodilution. British Journal of Pharmacology 128 (7), 1570–1576. Radziuk, J., 2000. Insulin sensitivity and its measurement: structural commonalities among the methods. The Journal of Clinical Endocrinology and Metabolism 85 (12), 4426–4433. Reaven, G.M., Hollenbeck, C., Jeng, C.Y., Wu, M.S., Chen, Y.D., 1988. Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37 (8), 1020–1024. Schoonjans, K., Staels, B., Auwerx, J., 1996. The peroxisome proliferators activated receptors (PPARs) and their effects on lipid metabolism and adipocytes differentiation. Biochimica et Biophysica Acta 1302 (2), 93–109. Scott, C., 2003. Diagnosis, prevention, and intervention for the metabolic syndrome. The American Journal of Cardiology 92 (1A), 35i–42i. Seip, M., Trygstad, O., 1996. Generalized lipodystrophy, congenital and acquired lipoatrophy. Acta Paediatrica, Supplementum 413, 2–28. Shepherd, J., Betteridge, J., Van Gaal, L., Panel, E.C., 2005. Nicotinic acid in the management of dyslipidaemia associated with diabetes and metabolic syndrome: a position paper developed by a European Consensus Panel. Current Medical Research and Opinion 21 (5), 665–682. Spiegelman, B.M., 1998. PPAR-gamma: adipogenic regulator and thiazolidinediones receptor. Diabetes 47 (4), 507–514. Storlien, L.H., Jenkins, A.B., Chisholm, D.J., Pascoe, W.S., Khouri, S., Kraegen, E.W., 1991. Influence of dietary fat composition on development of insulin resistance in rats: relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipids. Diabetes 40 (2), 280–289. Trayhurn, P., Beattie, J.H., 2001. Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. The Proceedings of the Nutrition Society 60 (3), 329–339. Uchiyama, K., Naito, Y., Hasegawa, G., Nakamura, N., Takahashi, J., Yoshikawa, T., 2002. Astaxanthin protects beta-cells against glucose toxicity in diabetic db/db mice. Redox Report: Communications in Free Radical Research 7 (5), 290–293. Vazquez, M., Silvestre, J., Prous, J., 2002. Experimental approaches to study PPAR gamma agonists and antidiabetic drugs. Methods and Findings in Experimental and Clinical Pharmacology 48 (8), 515–523.
G. Hussein et al. / Life Sciences 80 (2007) 522–529 Wang, Y., Lin, S., Liu, P., Cheung, B., Lai, W., 2004. Association between hematological parameters and metabolic syndrome components in a Chinese population. Journal of Diabetes and its Complications 18 (6), 322–327. WHO, 1999. Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications: Report of a WHO Consultation, Geneva, Switzerland: Department of Noncommunicable Disease Surveillance. World Health Organization. Xu, M., Xiao, S., Sun, Y., Zheng, X., Ou-Yang, Y., Guan, C., 2005. The study of anti-metabolic syndrome effect of puerarin in vitro. Life Sciences 77 (25), 3183–3196.
529
Yamauchi, T., Kamon, J., Waki, H., Imai, Y., Shimozawa, N., Hioki, K., Uchida, S., Ito, Y., Takakuwa, K., Matsui, J., Takata, M., Eto, K., Terauchi, Y., Komeda, K., Tsunoda, M.,Murakami,K., Ohnishi,Y., Naitoh, T., Yamamura, K., Ueyama, Y., Froguel, P., Kimura, S., Nagai, R., Kadowaki, T., 2003. Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis. The Journal of Biological Chemistry 278, 2461–2468.