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Chin Med J 2010;123(7):936-941

Original article
Calorie control increased vaspin levels of serum and periepididymal adipose tissue in diet-induced obese rats in association with serum free fatty acid and tumor necrosis factor alpha
WANG You-min, WANG Wen-ping, WANG Li-ping, L Qi-huan and ZHOU Xiao-hui Keywords: vaspin; diet-induced obese rats; pioglitazone; calorie control
Background Vaspin was recently identified as a novel adipokine that is predominantly secreted from adipose tissue and exerts insulin-sensitizing effects. This study was undertaken to elucidate the regulative effects of calorie control on the expression of vaspin and its potential mechanism. Methods Diet-induced obese Sprague Dawley (SD) rats were adopted as experimental models and accepted interventions of various ingestions and pioglitazone. Various differentiated stages of cultured 3T3-L1 cells were dealt with pioglitazone or TNF in vitro for 48 hours to further verify findings in animal experiments. Results The rats were successfully induced into an obese experimental model with hyperinsulinemia, hyperlipidemia, and increased serum free fatty acid and TNF by 12-week high-fat diet. It was found that depending on whether the rats were fed by a high-fat diet or a basal diet, there was extremely higher vaspin in the periepididymal fat pad than in subcutaneous adipose tissues by 16 weeks. Vaspin in sera and the periepididymal fat pad was much lower in rats with a high-fat diet than those with a basal diet (all P <0.05), but vaspin in subcutaneous fat tissues was prone to increase in rats with a high-fat diet. A 4-week calorie restriction or pioglitazone on the obese rats resulted in a partial recovery of vaspin levels in sera and periepididymal adipose tissues, especially the latter revealed a more obvious superiority and increased vaspin levels of subcutaneous adipose. Surprisingly, the treatment of 4-week high-fat diet on non-obese rats did not significantly depress vaspin of sera and periepididymal adipose tissues. However, it is unknown if re-feeding generated the effect on vaspin levels of obese and non-obese rats on sera or adipose tissues. The correlation analysis showed that vaspin levels of serum and periepididymal fat tissues were negatively correlated with serum FFA, TNF and insulin; meanwhile, there was a positive correlation between serum vaspin and vaspin of periepididymal fat tissues. Pioglitazone enhanced vaspin levels in cultured 3T3-L1 cells and supernatant in various differentiated stages, and this effect became more and more obvious along with the change of preadipocytes into mature fat cells. Administration of TNF caused suppression on vaspin expression in differentiated stages of 3T3-L1 cells. Conclusions The present data indicated that a long-term high-fat diet could induce obesity metabolic syndrome in SD rats and finally lead to lower vaspin of sera and periepididymal fat, while pioglitazone and chronic calorie-control ingestion could enhance the production of vaspin. It was undoubtedly demonstrated that vaspin expression was strongly associated with insulin sensitivity, serum FFA, and TNF. Chin Med J 2010;123(7):936-941

variety of adipocytokines and peptides (i.e. adiponectin, leptin, resistin, etc.) secreted from adipocytes have been considered to play key roles in obesity, insulin resistance, and type 2 diabetes.1-3 Visceral adipose tissue-derived serpin (vaspin) was recently identified as a member of the serine protease inhibitor (serpin) family, highly expressed in visceral adipose tissue when obesity and insulin levels peak in OLETF rats. Administration of recombinant vaspin was shown to improve glucose tolerance, insulin sensitivity in obese mice, and normalized altered expression of genes relevant to insulin resistance.4-6 In addition, it has been indicated that vaspin mRNA expression in human adipose tissue is regulated in a fat depot-specific manner.7 Because it was recently showed that serum vaspin concentration significantly increased approximately two-fold in normal glucose-tolerant, impaired glucose-tolerant, and type 2 diabetic subjects in response to the 4-week training program,8 the present study sought to explore the effects

of various ingestions on vaspin and possible mechanism of vaspin linked with insulin resistance. METHODS Materials Murine 3T3-L1 cells and fetal bovine serum (FBS) were purchased from the cell resources center at the Chinese
DOI: 10.3760/cma.j.issn.0366-6999.2010.07.031 Institute of Endocrinology & Metabolism, Anhui Medical University; Department of Endocrinology, First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China (Wang YM, Wang WP, Wang LP, L QH and Zhou XH) Correspondence to: Dr. WANG You-min, Institute of Endocrinology & Metabolism, Anhui Medical University, Hefei, Anhui 230022, China (Tel: 86-551-2923204. Fax: 86-5512922160. Email: youminwang@21cn.com) This work was supported by grants from the Natural Science Foundation of Anhui Province (070413080) and the Ministry of Education Foundation of Anhui Province (2006KJ089A).

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Academy of Sciences. 1-methyl-3-isobutylmethylx-anthine (IBMX), insulin (INS), and dexamethasone (DXM) were obtained from Sigma, USA. High-glucose DMEM was purchased from Gibcol BRL, USA. SD rats were purchased from an experimental animal company in Anhui province, China. Rabbit anti-vaspin monoclonal antibody was purchased from Phoenix Bioteth Co. Ltd. Donkey anti-rabbit IgG-HRP conjugate, antibody dilution, and lysis solution were obtained from Beyotime, China. SuperSignal West Femto Maximum Sensitivity Substrate was purchased from Pierce Biotechnology, USA. Animal breed and experimental protocol The 6-week old male Sprague-Dawley (SD) rats (15317) g were maintained in individual cages under controlled temperature (21C23C), light (12-hour light, 12-hour dark), and humidity (555)% conditions with access to food and water ad libitum. After a 1-week acclimation period, the rats were randomly divided into two groups given the basal diet (n=40) and the high fat diet (n=40, containing 50% normal chow, 12% lard, 5% cane sugar, 8% milk powder, 5% peanut, 10% heneggs, 3% sesame oil and 2% common salt) for 12 weeks. Then the group of rats following the basal diet was randomly divided into three subgroups: keeping basal diet (KBD, n=20), converting into high-fat diet (BDH, n=10), or intragastric administration of pioglitazone (BDP, n=10), and fed for another 4 weeks, respectively. The group with the high fat diet was also randomly divided into three subgroups: keeping high fat diet (KHD, n=20), changing into basal diet (HDB, n=10), and intragastric administration of pioglitazone (HDP, n=10) for 4 weeks. Except part of the KBD and the KHD subgroup, rats received refeeding after fasting 8 hours. The other rats were kept in fasting for 8 hours before anesthetized using 10% chloral hydrate and sacrificed. The blood from the inferior vena cava of the rats was obtained to measure the biochemical characteristics. Then, subcutaneous and periepididymal adipose tissues were collected from individual donors, and those samples were snap-frozen in liquid nitrogen for later use. Animal experiments were conducted in accordance with the Internal Animal Care and Use Committee of Anhui Medical University and complied with the Guide for the Care and Use of Laboratory. 3T3-L1 cell culture and experimental protocol 3T3-L1 cells were grown to confluence in a Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS in humidified 5% CO2 at 37C. Differentiation of the 3T3-L1 cells was induced as described previously. In brief, after 2 days of confluence, the 3T3-L1 cells were incubated with DMEM containing 10% FBS, 3-isobutyl1-methylxanthine (0.5 mmol/L), dexamethasone (0.1 mol/L), and insulin (10 g/ml) for 48 hours. the culture medium was changed every other day with DMEM containing 10% FBS and insulin (10 g/ml). At day 8, at least 95% of the differentiated 3T3-L1 cells exhibited intracellular lipid droplets; it was then inferred that the 3T3-L1 preadipocytes were induced into mature

adipocytes. During differentiation, the 3T3-L1 cells were treated with vehicle, pioglitazone (100 mol/L), TNF (100 ng/ml) at 0-, 2-, 4-, 6-, and 8-day intervals for 48 hours, respectively, and each experiment was repeated 6 times. Then the cells and serum-deprived supernatant were harvested and pooled for vaspin expression analysis. Protein extraction and Western blotting analysis for vaspin Protein from the cultured cell and fat tissues was extracted with a lysis buffer, and then equal amounts of total protein from each sample were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose filter using an electroblotting apparatus. The membrane was blocked with blocking solution for 3 hours at room temperature, and then incubated overnight at 4C with rabbit anti-vaspin (diluted 1: 300 in western antibody dilution). After 10-minute washing for three times in PBST, the membranes were incubated for 1 hour at room temperature with a horseradish peroxidase-conjugated anti-rabbit secondary antibody at 1:1000 in blocking solution. Signal was developed using West Pico chemiluminescence substrate (Pierce, USA). Bands were then quantified by scanning densitometry using protein gel graph analyzing system. To confirm equal loading, the same membranes were stripped by stripping buffer and distilled water for 5 minutes respectively at room temperature. Then -actin was used as a housekeeping protein, and was determined following the same procedure as mentioned above, using a specific anti-actin rabbit polyclonal antibody at 1:280 and a horseradish peroxidase-conjugated anti-rabbit secondary antibody at 1:2000 in blocking solution. Biochemical analyses The concentrations of serum free fatty acid, TNF, lipid profile, and fasting glucose were determined enzymatically using diagnostic kits. Fasting insulin was assayed by a rat-specific radioimmunoassay as described by the supplier (Linco, UAS). Statistical analysis All the statistical analyses were executed using the SPSS program, version 13.0, for Windows. The skewed variables were transformed into natural logarithms. All data are expressed as mean standard deviation (SD). Comparisons between groups were carried out using one way analysis of variance (ANOVA) and Students paired t test. The correlation analysis was tested using the Pearson correlation model t test. A value of P <0.05 (two-tailed) was regarded as statistically significance. RESULTS Effects of various regimens and pioglitazone on metabolic parameters The body weight of rats fed with the high fat diet gradually increased with prolonged time and achieved a

938 Table 1. Metabolic characteristics of SD rats at 16 weeks

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Groups BW (g) EAT (g) FBG (mmol/L) FINS (mIU/L) TG (mmol/L) TCH (mmol/L) TNF (mmol/L) KBD 456.916.0 3.470.34 5.010.18 47.517.6 0.530.04 1.230.05 23.32.1 BDH 478.317.7 4.370.66 5.130.27 70.228.1* 0.650.07 1.310.08 26.92.6 BDP 471.518.7 3.560.52 4.970.22 42.514.7 0.520.06 1.210.06 21.52.3 KHD 555.715.9* 7.190.72* 6.140.34* 84.939.7* 0.910.10* 1.480.09* 41.12.8* HDB 513.121.8 5.490.59 5.400.29 62.723.8 0.800.08 1.400.04 33.84.3 HDP 564.418.9 6.900.65 5.100.27 49.916.4 0.820.04 1.360.05 23.93.1 * The data of rats without fasting were not included in the table. P <0.05 vs. KBD group; P <0.05 vs. KHD group, P <0.05 vs. BDP group. BW: periepididymal adipose tissue; FFA: free fatty acid; TG: triglyceride; TCH: total cholesterol: FBG: fasting blood glucose: FINS: fasting blood insulin.

FFA (mol/L) 411.624.8 502.343.2* 407.521.3 640.027.3* 558.638.6 484.131.1 body weight; EAT:

Figure 1. The effects of various regimens and pioglitazone on vaspin in adipose tissues and sera at 16 weeks. A: The data were enrolled from rats with keeping 8-hours fasting ahead of sacrifice. B: The data of rats fed by keeping the basal diet were displayed by means of comparing 6-hours fasting with foodintake state before being executed. C: The data of rats fed by keeping high-fat diet were shown to compare the differences of vaspin levels in fasting and foodintake state. Data are presented as meanSE for each group (n=15). Serum vaspin level was presented by stripe densitometry minified 1000. *P <0.05 vs. KBD group; P <0.05 vs. KHD group. KBD: Rats were fed by keeping basal diet. BDH: Rats were fed from the basal diet into high-fat diet in the last 4 weeks. BDP: Rats were fed by keeping basal diet and given intragastric administration of pioglitazone in the last 4 weeks. KHD: Rats were fed by keeping high fat diet. HDB: Rats were fed from high fat diet into basal diet in the last 4 weeks. HDP: Rats were fed by keeping high fat diet and given intragastric administration of pioglitazone in the last 4 weeks. KBDF: Rats fed by keeping the basal diet are in foodintake state before being executed. KHDF: Rats fed by keeping high-fat diet are in foodintake state before being executed. SAT: subcutaneous adipose tissue. EAT: periepidydimal adipose tissue.

20% of body weight more than rats with the basal diet until 12 weeks ((536.232.8) g vs. (442.829.2) g, P=0.038). By the end of the experiment, some characteristics of metabolic syndrome, besides body weight, in rats consuming only high fat chow were increased serum free fatty acid and TNF, fasting glucose and insulin, total triglyceride and cholesterol, and the marked periepididymal fat accumulation (Table 1). After some rats were reared by converting from the high fat diet to the basal diet for 4 weeks, the parameters mentioned above were partly reduced when compared with the rats that remained on the high fat diet. Pioglitazone decreased the levels of free fatty acid and TNF, fasting glucose and insulin (P <0.05 for all) but not triglyceride or total cholesterol. In contrast, when some rats were changed from the basal diet to the high diet for 4 weeks, the parameters mentioned above were augmented to some extent when compared with rats that remained on the basal diet, especially in serum insulin and FFA. Effects of various regimens and pioglitazone on vaspin in sera and adipose tissues Whether the rats maintained the high fat diet or the basal diet for 16 weeks, vaspin was unexceptionally higher in periepididymal fat tissues than in subcutaneous adipose tissues. It was also found that vaspin in sera and periepididymal adipose tissues was much lower in rats with the high fat diet than with the basal diet (all P <0.05), but vaspin in subcutaneous adipose tissues was prone to increase in rats with the high fat diet. When the rats

underwent the change from the high fat diet to the basal diet for 4 weeks, the decline of vaspin level in sera and periepididymal adipose tissues were partly reversed. When the rats underwent the reverse, from the basal diet to the high fat diet, no significant decrease of vaspin was found whether in sera, periepididymal, or subcutaneous adipose tissues compared with rats with keeping the basal diet. In rats that maintained the high fat diet, the 4-week treatment of pioglitazone increased the vaspin level in sera, periepididymal, and subcutaneous adipose tissues, especially in periepididymal fat tissues, close to the level of rats maintaining the basal diet. Interestingly, regardless of the basal diet or the high fat diet, no significant difference was shown in the vaspin level in sera, periepididymal, and subcutaneous adipose tissues in between the fasting and the intaking state (Figures 1 and 2) .

Figure 2. The effects of various regimens and pioglitazone on vaspin. Pictures were representatives of vaspin expression detected by Western immunoblot analysis in adipose tissues and sera at 16 weeks.

Correlation analysis of vaspin expression with anthropometric and metabolic parameters In order to evaluate the correlation of vaspin and

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metabolic characteristics, the data of rats fed with the high fat or/and the basal diet, but not those that received an intervention of pioglitazone, were used to analyze the correlation. The results showed that serum fasting vaspin negatively correlated with body weight, serum FFA, TNF, insulin, and triglyceride, but positively with vaspin level in periepididymal fat tissues. Meanwhile, the vaspin level of periepididymal fat tissues also negatively correlated with the weight of periepididymal fat, serum FFA and TNF. Whereas, no significant correlation was obtained between vaspin in subcutaneous adipose and the metabolic parameters. (Table 2).
Table 2. Correlation of vaspin with anthropometric and metabolic parameters
Vaspin in sera Vaspin in EAT Vaspin in SAT r values P values r values P values r values P values BW (g) 0.509 0.000 0.511 0.000 0.201 0.246 EAT (g) 0.752 0.000 0.750 0.000 FBG (mmol/L) 0.337 0.055 0.297 0.093 0.110 0.544 FINS (mIU/L) 0.390 0.025 0.416 0.039 0.009 0.955 TG (mmol/L) 0.357 0.042 0.343 0.051 -0.223 0.212 TCH (mmol/L) 0.121 0.494 0.120 0.499 0.097 0.586 TNF (mmol/L) 0.714 0.000 0.815 0.000 -0.192 0.320 FFA (mol/L) 0.433 0.012 0.449 0.009 -0.029 0.861 Vaspin in SAT 0.287 0.102 Vaspin in EAT 0.877 0.000 For this analysis, Pearson correlation model was used. P <0.05 (two-tailed) was considered statistically significant. Characteristics

DISCUSSION The metabolic syndrome is defined by the co-occurrence of intra-abdominal obesity, atherogenic dyslipidemia, raised blood pressure (BP), insulin resistance and/or glucose intolerance, and a proinflammatory state. The present data demonstrate that this model induced by a high fat diet exhibits many of these features, such as dyslipidemia, increased TNF and FFA concentration, decreased insulin sensitivity calculated by elevating the fasting plasma glucose and insulin. In particular, the marked fat in the visceral white adipose tissue accumulated significantly more in the three-month high fat diet group than in the basal diet group. The fat source of the high fat diet in this study consisted of saturated fatty acids derived from animal fats, which have been known to have deleterious effects on health; they have been specifically implicated in activating the toll-like receptor-4, leading to the expression of inflammatory gene products.9,10 Although it is still unclear how excess visceral fat triggers metabolic syndrome, many data have clearly indicated that a high fat diet induces visceral fat accumulation preceding insulin resistance and increases visceral adipose tissue mass associated with a higher prevalence of insulin resistance, type 2 diabetes, and the risk of cardiovascular disease.11-13 Visceral fat depot-specific secretion of adipokines might, at least in part, explain the adverse effects of intra-abdominal fat accumulation. Vaspin was isolated from the visceral adipose tissue of OLETF rats with abdominal obesity, insulin resistance, hypertension, and dyslipidemia. Glucose tolerance and insulin sensitivity was improved in obese mice by administration of recombinant vaspin.4,5 It is well known that a favorable lifestyle can increase insulin sensitivity, and decrease risks of diabetes and coronary heart disease associated with insulin resistance. A previous study in humans revealed that vaspin correlated with metabolic disorders,14,15 and training improved insulin sensitivity and significantly increased vaspin serum concentration.8 Nevertheless, it is still indefinite whether meals can

Effects of TNF- or/and pioglitazone on 3T3-L1 cells and corresponding supernatant Vaspin expressed in 3T3-L1 preadipocytes at low levels but was still detectable, and only slightly increased in cultured adipocytes and corresponding supernatant until the cell became mature (P >0.05 for all). After the treatment of pioglitazone on the fully differentiated 3T3-1 cells for 48 hours, the vaspin concentration of cells and supernatant were approximately enhanced by 58% compared with the control experiment (P <0.05 for all). However, the 48-hours intervention of TNF- inhibited vaspin protein expression in differentiated stages of 3T3-L1 cells reduced vaspin by 76% in full-grown cells and 73% in the cultured supernatant (Figure 3).

Figure 3. Vaspin levels in 3T3-L1 cells and supernatant. The curves represented mean changes of vaspin levels in 3T3-L1 cells and supernatant according to vehicle, pioglitazone or TNF treatment on cultured cells in vitro during differentiation (C and D). Vaspin level in cultured supernatant was presented by stripe densitometry minified 1000. Pictures were representatives of vaspin expression detected by Western blotting analysis after intervention of vehicle, pioglitazone or TNF (A and B) on cultured cells. *P <0.05 vs. NC group. NC: normal control. PIO: pioglitazone.

940

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regulate vaspin expression thus far. In the present study, vaspin concentrations in sera and periepididymal fat tissues were decreased in response to the 16-week high fat diet, which was compared with the basal diet; this decrease was accompanied by increased FFA and TNF, total triglyceride and cholesterol, fasting plasma glucose and insulin, and periepididymal fat weight. Vaspin level in sera had a negative correlation with serum FFA, TNF, fasting insulin and total triglyceride, and periepididymal fat weight, but a positive correlation with vaspin of periepididymal adipose tissue. Simultaneously, the vaspin level of periepididymal fat tissue negatively correlated with the weight of periepididymal adipose, serum FFA, and TNF. It seemed that these results displayed a paradoxical manifestation that vaspin levels decreased as body weight and visceral adipose increased even though vaspin was secreted from adipose tissue. However, it was reasonably inferred that descending vaspin was a result of worsened insulin resistance and elevated coherent nocuous adipokines induced by the high fat diet. In addition, it was not shown that temporary refeeding significantly had an effect on vaspin level of sera and periepididymal fat tissues of rats in the present study. It was implied that vaspin, unlike other adipocytokines, such as leptin which acts as energy ingestion sensor, participated in retaining homeostasis by means of visceral fat depot-specific secretion. After the rats were reared from the high fat diet to the basal diet for 4 weeks, vaspin in sera and periepididymal adipose tissues ascended to a great extent. Furthermore, these changes of vaspin in the rats kept a sizeable syntropy with serum FFA and TNF at 16 weeks. Surprisingly, although vaspin in subcutaneous adipose tissue was prone to increase in rats with the high fat diet, no significant correlation was obtained between vaspin of subcutaneous adipose and metabolic parameters. This strongly reinforced the presumption that decreased serum vaspin had a direct relationship with worsened insulin resistance due to visceral obesity induced by a chronic high fat diet. The primary study showed that vaspin mRNA was abundantly and exclusively expressed in visceral white adipose tissues at the age of 30 weeks, when OLETF rats reach their peak body weight. It has been thought that vaspin may be the compensatory molecule in the pathogenesis of the metabolic syndrome.4,5 In contrast, the present study revealed that vaspin levels in sera and periepididymal adipose tissues were significantly decreased in response to the 16-week high fat diet, although an intervention of the 4-week high fat diet on non-obese control rats had not brought out significantly lower vaspin levels in sera and periepididymal adipose tissues. Unfortunately, we had not observed the impact of age and body weight on vaspin expression of rats in series. It has been reported that PPAR-gamma agonist, including pioglitazone, which is generally acknowledged as an insulin sensitizer, can cause preadipocytes to differentiate into mature fat cells and induce key enzymes involved in lipogenesis.16-18 The present study discovered that

pioglitazone augmented the vaspin concentration in sera and adipose tissues, simultaneously reduced serum FFA and TNF, and improved insulin sensitivity. That is to say, ameliorating insulin resistance would be followed by the elevating vaspin in sera and fat tissues. In vitro, our study showed that vaspin could be expressed in 3T3-L1 preadipocytes and differentiated adipocytes, and secreted into the supernatant, which indicated that vaspin was a true adipokine and secretory protein. After various differentiated stages of 3T3-L1 cells were dealt with pioglitazone for 48 hours, vaspin levels in cultured cells and supernatant were enhanced to some degree, and the effect of pioglitazone on vaspin expression became more and more obvious along with the change of preadipocytes into mature fat cells. These results showed that the administration of pioglitazone produced inverse changes on vaspin levels of sera and fat tissues with serum FFA and TNF in vivo, and had a greater effect on vaspin expression in mature fat cells in vitro. It indicated that the effects of pioglitazone on vaspin expression are closely linked with its pharmacological mechanisms, such as facilitating preadipocyte differentiation and decreasing serum FFA and TNF. In contrast to pioglitazone, many studies have demonstrated that TNF plays a potential role in insulin resistance, both in cultured adipocyte and whole-animal models.19-21 Our study discovered that the administration of TNF caused suppressed vaspin expression in fully differentiated stages of 3T3-L1 cells in vitro. In addition, after diet-induced obese rats were intervened by restricting calorie ingestion for 4 weeks, vaspin levels of sera and visceral fat tissues were retrieved along with improving insulin sensitivity and decreaseing serum FFA and TNF concentration. Thus, it is conceivable that measures of boosting insulin sensitivity, including insulin sensitizer, training and restricting calorie ingestion can up-regulate vaspin expression in fat tissues, especially in the visceral adipose tissue. However, precise supplementary studies are needed to expound whether alimentary control accelerates or decelerates the production of vaspin and its mechanism of regulation in humans. According to our present findings, these data supported the idea that a chronic high fat diet induces the obesity metabolic syndrome in SD rats, and then leads to lower vaspin of sera and periepididymal fat tissues, but pioglitazone and chronic control-calorie ingestion can enhance the production of vaspin. Additional studies are definitely necessary to identify whether alimentary control can attain the same benefit to vaspin in humans and to trace the curve of vaspin fluctuation following an extension of breeding time in rats with a high fat diet. Some studies4-6 and present data have demonstrated that vaspin is a novel adipokine that has insulin sensitizing effects and suggest that vaspin can play an important role in the amelioration of obesity and metabolic disorders. Thus, it seems appropriate to use serum vaspin levels as a marker to evaluate obese and diabetic patients for metabolic disorders and medications interfering with

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941 12. Frayn KN. Visceral fat and insulin resistance-causative or correlative? Br J Nutr 2000; 83 (Suppl 1): S71-S77. 13. Wajchenberg BL. Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr Rev 2000; 21: 697-738. 14. Suleymanoglu S, Tascilar E, Pirgon O, Tapan S, Meral C, Abaci A. Vaspin and its correlation with insulin sensitivity indices in obese children. Diabetes Res Clin Pract 2009; 84: 325-328. 15. Gulcelik NE, Karakaya J, Gedik A, Usman A, Gurlek A. Serum vaspin levels in type 2 diabetic women in relation to microvascular complications. Eur J Endocrinol 2009; 160: 65-70. 16. Sharma AM, Staels B. Peroxisome proliferator-activated receptor gamma and adipose tissueunderstanding obesity-related changes in regulation of lipid and glucose metabolism. J Clin Endocrinol Metab 2007; 92: 386-395. 17. Campbell IW. The clinical significance of PPAR gamma agonism. Curr Mol Med 2005; 5: 349-363. 18. Laplante M, Festuccia WT, Soucy G, Glinas Y, Lalonde J, Berger JP, et al. Mechanisms of the depot specificity of peroxisome proliferator-activated receptor gamma action on adipose tissue metabolism. Diabetes 2006; 55: 2771-2778. 19. Hivert MF, Sullivan LM, Fox CS, Nathan DM, DAgostino RB Sr, Wilson PW, et al. Associations of adiponectin, resistin, and tumor necrosis factor-alpha with insulin resistance. J Clin Endocrinol Metab 2008; 93: 3165-3172. 20. Ishizuka K, Usui I, Kanatani Y, Bukhari A, He J, Fujisaka S, et al. Chronic tumor necrosis factor-alpha treatment causes insulin resistance via insulin receptor substrate-1 serine phosphorylation and suppressor of cytokine signaling-3 induction in 3T3-L1 adipocytes. Endocrinology 2007; 148: 2994-3003. 21. Cheng YL, Li L, Yang GY, Shi SC, Chen Y, Zhu W, et al. Effects of tumor necrosis factor-alpha induced insulin resistance on adipose triglyceride lipase in mice. Natl Med J China (Chin) 2008; 88: 2417-2421.

serum vaspin levels.

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(Received July 11, 2009) Edited by WANG Mou-yue and LIU Huan