AIM: To evaluate the potential acylation stimulating protein (ASP) resistance in both adipocytes and preadipocytes under the conditions by which insulin resistance is produced by the stimulation of free fatty acids (FFA), and to explore the mechanism of ASP resistance on post-receptor level. METHODS: 3T3-L1 preadipocytes were induced to differentiate. Then the cells were treated with oleate or palmitate at concentration of 0 mmol/L (FFA-free DMEM/F12), 0.125 mmol/L, 0.5 mmol/L or 1.0 mmol/L overnight. Glucose transport was assessed by [~3H] 2-deoxyglucose uptake to evaluate insulin resistance and ASP resistance. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with ASP at concentration of 5.0 μmol/L for 4 h, then the cell proteins were extracted, and the expressions of guanine nucleotide binding protein beta (Gβ), guanine nucleotide-binding protein alpha-q/11(Gαq/11), phosphorylated-protein kinase Cα (p-PKCα) and phosphorylated-protein kinase Cζ (p-PKCζ) were measured by Western blotting. RESULTS: Both adipocytes and preadipocytes were responsive to ASP. ASP stimulation increased glucose transport by 198% in adipocytes and by 287% in preadipocytes (P<0.01 vs PBS). FFA at concentration of 0.125 mmol/L did not change ASP-stimulated glucose transport significantly, but high dose of oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% (P<0.05 for oleate) and 34% (P<0.05 for palmitate) at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes, glucose uptake rates were decreased by 43% (P<0.05 for oleate) and 62% (P<0.01 for palmitate) at 1 mmol/L FFA. Effects were comparable to those obtained with insulin. After overnight incubation with oleate or palmitate in adipocytes and preadipocytes, Gβ, Gαq/11, p-PKCα and p-PKCζ were downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. At concentration of 1.0 mmol/L, oleate inhibited the expressions of ASP-induced Gβ, Gαq/11, p-PKCα and p-PKCζ in adipocytes by 47%, 44%, 39% (P<0.05, P<0.01) and 20% (P>0.05), respectively. Palmitate also effectively blocked the expressions of ASP (at concentration of 1.0 mmol/L)-induced Gβ, Gαq/11, p-PKCα and p-PKCζ by 50%, 43%, 44% and 43% (P<0.05, P<0.01) in adipocytes. In preadipocytes, oleate only inhibited ASP-induced p-PKCα and p-PKCζ significantly by 39% and 19%, respectively (P<0.05). However, overnight exposure of 3T3-L1 preadipocytes to 1 mmol/L palmitate leaded to 45%, 50%, 52% and 21% (P<0.05, P<0.01) inhibition of ASP-induced expressions of Gβ, Gαq/11, p-PKCα and p-PKCζ, respectively. CONCLUSION: Oleate and palmitate inhibit ASP-mediated stimulation of glucose transport both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance under the condition of insulin resistance induced by FFA in a cellular model. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and obesity phenotype.

AIM: To study the expression of three kinds of transcriptional factors PPAR?, C/EBP? and C/EBP ?mRNA during differentiation in 3T3-L1 preadipocytes induced by acylation stimulating protein. METHODS: There were three groups in the study divided by the difference differentiation inducer: (1) control group: incubating the cells without any inducer; (2) IBMX+DEX group: incubating the cells with IBMX and DEX; (3) ASP group: incubating the cells with ASP, IBMX and DEX. The insulin of the typical hormone cocktail method was taken place by ASP. 3T3-L1 preadipocytes were induced to differentiate by 50 mg/L ASP+0.5 mol/L IBMX+1.0 ?mol/L DEX. The cells were harvested on the first day, second day,4th day, 6th day and 8th day after differentiation, then the total RNA of these cells were abstracted. The transcription factors PPAR?, C/EBP?, and C/EBP? mRNA expressions were assayed by RT-PCR. RESULTS: (1) During the differentiation induced by ASP group, PPAR? mRNA expression in the 3T3-L1 cells were very low on the first day after inducing differentiation. The expression was increased lightly on the second and 4th day after inducing differentiation, and it was kept on the high level on the 6th and 8th day after induction. The C/EBP? mRNA was expressed at low level on the first day after inducing differentiation. It was increased significantly on the second day and decreased significantly on the 4th day after induction. C/EBP? mRNA was not be detected on the 6th and 8th day after induction. The expression of C/EBP? mRNA was low on the first day after inducing differentiation. It was increased on the second and 4th day after induction and it was kept on high level on the 6th and 8th day after induction. (2) During the differentiation induced by IBMX+DEX group, PPAR?, C/EBP? and C/EBP? mRNA expressions were increased at the beginning of differentiation, but the levels of expression were lower than those in ASP group. CONCLUSION: The sequential expression of these transcription factors induced by ASP may be the important mechanism for the role of ASP to induce the preadipocytes to differentiate.

Objective To study the relationship of plasma aeylation stimulating protein (ASP) with complement C3, C-reactive protein (CRP) and blood lipid levels in women with polyeystic ovary syndrome (PCOS). Methods Thirty-four patients with PCOS were divided into two groups: obese PCOS group [body mass index (BMI)≥25 kg/m2] and non-obese PCOS group (BMI<25 kg/m2). 41 age-matched non-PCOS women were also divided into two groups: simply obese group (BMI≥25 kg/m2) and non-obese control group (BMI <25kg/m2). Plasma ASP in the 4 groups was detected by enzyme linked immunosorbent assay (ELISA) method.Complement C3 and CRP were determined by immunoturbidimetrie assay. Plasma free fatty acid (FFA)concentration was determined by colorimetric enzymatic assay, plasma triglycerides (TG) by GPO-PAP method and total cholesterol (TC) by COD-PAP method. Results The plasma ASP were significantly increased in the obese PCOS group, the non-obese PCOS group and the obese group as compared with the control group [(36.4±10.9,34.8±9.9, 35.1±14.0, 24.8±7.8) nmol/L, respectively, all P<0.05]. The concentrations of complement C3 were significantly higher in the obese PCOS group and the obese group than that in the control group [(2.2±1.2,2.5±1.5, 1.1±0.7) g/L, respectively, bothP <0.05]. The concentrations of CRP were significantly increased in the obese PCOS group, non-obese PCOS group and obese group as compared with the control group [(32.1±29.2, 30.0±24.8, 23.8±5.5, 7.5±4.8)mg/L, respectively, all P<0.05]. Univariate analysis showed that both plasma ASP and C3 were positively correlated with BMI, CRP, FFA and TG. CRP was positively correlated with BMI, FFA, TG and TC. Conclusion Plasma ASP, C3 and CRP levels in women with PCOS axe increased. They are strongly associated with disturbed lipid metabolism. The lack of association between ASP and complement C3 suggests that the conversion of C3 to ASP may be affected by other factors.

AIM:Heart failure is characterized by immune activation leading to production and release of proinflammatory cytokines .Inter-leukin 17A (IL-17A) is a proinflammatory cytokine and multiple lines of evidence from animal and human studies suggest crucial roles of IL-17A in heart failure.Therefore, we investigated whether common polymorphisms of genes IL17A and IL17RA (coding interleukin 17 receptor A) gene contribute to genetic predisposition to heart failure and adverse clinical outcomes associated with it .METHODS AND RESULTS:A total of 1713 adults patients with congestive heart failure and 1713 age-and sex-matched controls were genotyped for promoter SNPs, rs2275913 and rs8193037 in IL17A and rs4819554 in IL17RA, to assess the relationship between individual SNPs and the risk of congestive heart failure .Results showed that rs8193037 in IL17A was associated with the risk of congestive heart failure (P<0.01) after adjustment for multiple cardiovascular risk factors including age , sex, smoking status, diabetes, hypertension and dyslipidemia.This association was evident in both ischemic and non-ischemic heart failure (P<0.05).Furthermore, prospective fol-low-up of 12.7 months for the occurrence of adverse clinical outcomes showed that rs 4819554 in IL17RA was significantly associated with cardiovascular mortality (P<0.05) after adjustments for multiple cardiovascular risk factors and New York Heart Association functional class.CONCLUSION:This study demonstrated associations of rs8193037 in the promoter of IL17A with the risk of conges-tive heart failure, and of rs4819554 in the promoter of IL17RA with the risk of cardiovascular mortality in patients with congestive heart failure.These data lend further support to the notion that immune activation and genetic polymorphisms contribute to heart failure path -ogenesis and progression .

This study examined the role of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6) and cystatin C in ischemic stroke complicating atrial fibrillation (AF) and the relationship of systemic inflammation with this disease in order to identify AF patients who are at high risk of stroke and need optimal anticoagulant therapy. A total of 103 AF patients, simple (n=75) or complicated by ischemic stroke (n=28), and 112 control subjects were recruited. IL-6 level was detected by using enzyme linked immunosorbent assay. Cystatin C and hsCRP levels were measured by means of a particle-enhanced immunonephelometric assay. The results showed that the AF patients had higher levels of hsCRP (P=0.004), IL-6 (P=0.000), and cystatin C (P=0.000) than control subjects. Plasma hsCRP level was increased in the AF patients with ischemic stroke as compared to the patients with simple AF (P=0.036). The AF patients who had the level of hsCRP exceeding 3.83 mg/L were at a higher risk than those with hsCRP level lower than 3.83 mg/L (P=0.030). After adjusting for other factors, cystatin C remained positively associated with IL-6 (r=0.613) and hsCRP (r=0.488). It was concluded that hsCRP is positively correlated with ischemic stroke complicating AF and may be a risk factor independent of other risk factors for AF. Elevated cystatin C level is also indicative of the increased risk of AF.

OBJECTIVETo explore the role of adipokines including insulin, resistin, leptin, adiponectin, acylation stimulating protein (ASP) and complement C3 (C3) in various types of obesity (peripheral obesity, abdominal obesity and mixed obesity) in Chinese children and adolescents, and their relationships with body size and pubertal development.

METHODSChildren and adolescents (n=3 508) aged 6 to 18 years, with 1 788 boys and 1 720 girls were assessed for body mass index, waist circumference, pubertal development, blood insulin, resistin, leptin, adiponectin, ASP and C3 levels. Three types of obesity [peripheral obesity (n=43), abdominal obesity (n=473), mixed obesity (n=1 187)] and non-obese control (n=1 805) were defined with combined use of Chinese body mass index and waist circumference criteria.

RESULTSSerum resistin, leptin and adiponectin levels were higher in girls than those in boys (all P<0.01). Insulin and leptin increased and adiponectin decreased across five Tanner stages in both girls and boys (all P<0.001), while ASP changed only in girls (P<0.001) and C3 only in boys (P<0.001). Insulin, leptin and ASP were higher, but adiponectin was lower in all three types of obesity vs. the non-obese control (all P<0.05). The greatest abnormalities of all six adipokines were found in the mixed obesity group. With inclusion of body mass index and waist circumference in simultaneous regression analyses, both body size indices were independently and significantly correlated with insulin, leptin and adiponectin after age and gender adjustment. Compared with waist circumference, the body mass index was stronger in interpreting insulin, leptin, adiponectin and ASP levels, whereas it was weaker in explaining variance of plasma C3.

CONCLUSIONObese children have a worse metabolic profile with high insulin, resistin, leptin, ASP and C3, and low adiponectin levels. The adipokine profile in mixed obesity is worse than that in peripheral or abdominal obesity. Identification of obese subjects with a malignant adipokine profile using a combination of body mass index and waist circumference is important for the prevention of obesity-related disease.

Adipokines ; blood ; Adolescent ; Child ; China ; Cross-Sectional Studies ; Female ; Humans ; Male ; Obesity ; blood

This study examined the role of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6) and cystatin C in ischemic stroke complicating atrial fibrillation (AF) and the relationship of systemic inflammation with this disease in order to identify AF patients who are at high risk of stroke and need optimal anticoagulant therapy. A total of 103 AF patients, simple (n=75) or complicated by ischemic stroke (n=28), and 112 control subjects were recruited. IL-6 level was detected by using enzyme linked immunosorbent assay. Cystatin C and hsCRP levels were measured by means of a particle-enhanced immunonephelometric assay. The results showed that the AF patients had higher levels of hsCRP (P=0.004), IL-6 (P=0.000), and cystatin C (P=0.000) than control subjects. Plasma hsCRP level was increased in the AF patients with ischemic stroke as compared to the patients with simple AF (P=0.036). The AF patients who had the level of hsCRP exceeding 3.83 mg/L were at a higher risk than those with hsCRP level lower than 3.83 mg/L (P=0.030). After adjusting for other factors,cystatin C remained positively associated with IL-6 (r=0.613) and hsCRP (r=0.488). It was concluded that hsCRP is positively correlated with ischemic stroke complicating AF and may be a risk factor independent of other risk factors for AF. Elevated cystatin C level is also indicative of the increased risk of AF.

OBJECTIVETo analyze the characteristics of a new clinical syndrome, including throat infection, neck spinal disease, chest pain and cardiac response.

METHODSA total of 165 patients with above mentioned symptoms admitted to Tongji hospital from 2003 to 2005 were included in this study and underwent further medical history inquiry, physical examination and laboratory tests. Eighty-five healthy subjects served as controls. Serum myocardial auto-antibodies against beta(1)-adrenoceptor, alpha-myosin heavy chain, M(2)-muscarinic receptor and adenine-nucleotide translocator were detected, inflammatory cytokines, high sensitivity C-reaction protein, serum antibodies against Coxsackie virus-B, cytomegalovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae were determined and lymphocyte subclasses were assayed by flow cytometry.

RESULTSAll patients had a complex of four symptoms or tetralogy: (1) persistent throat or upper respiratory tract infection; (2) neck pain; (3) chest pain; (4) chest depression or dyspnea, some of them with anxiety. Anti-myocardial auto-antibodies (AMCA) were present in all patients vs. 8% in controls. TNF-alpha, IL-1 and IL-6 were significantly higher in patients than controls (P < 0.01). CD3(+) and CD4(-)CD8(+) lymphocytes were significantly higher and CD56(+) lymphocytes lower in patients than those in controls (P < 0.01). The ratios of serum pathogen antibodies positive against Coxsackie virus-B, cytomegalovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae were all significantly higher in patients than in controls.

CONCLUSIONSThese data led to identification of a persistent respiratory infection-related clinical syndrome, including persistent throat infection, neck spinal lesion, rib cartilage inflammation, symptoms of cardiac depression and dyspnea with or without anxiety.

Adolescent ; Adult ; Aged ; Anxiety ; diagnosis ; Case-Control Studies ; Chest Pain ; diagnosis ; Female ; Heart Diseases ; diagnosis ; Humans ; Inflammation ; Male ; Middle Aged ; Neck Pain ; diagnosis ; Respiratory Tract Diseases ; diagnosis ; microbiology ; Spinal Diseases ; diagnosis ; Syndrome ; Young Adult

BACKGROUNDThe levels of long-term elevated serum or intracellular free fatty acid (FFA) induce insulin resistance associated with central obesity. The insulin-mimetic protein visfatin is preferentially produced by visceral adipose tissues and has been implicated in obesity and insulin resistance. To identify that FFA is capable of inducing insulin resistance and to clarify the role of FFA on visfatin, we examined the effect of monounsaturated FFA oleate (C18:1) and saturated FFA palmitate (C16:0) on glucose transport and visfatin gene expression in cultured 3T3-L1 adipocytes or preadipocytes.

METHODSFFA-free DMEM/F12, 0.125 mmol/L, 0.5 mmol/l and 1.0 mmol/L oleate or palmitate was added to cultured 3T3-L1 adipocytes or preadipocytes and incubated overnight. Glucose transport was assessed as (3)H-2-deoxy-glucose uptake. Total RNA was extracted and subjected to RT-PCR for the measurement of visfatin mRNA levels. Statistical comparisons between control group and other groups were performed with the two-tailed paired t test, and one-way ANOVA was used to compare the mean values among the groups.

RESULTSInsulin increased specific membrane glucose transport in 3T3-L1 preadipocytes. Upregulation was evident from 15 minutes to 1 hour exposure to insulin. However, after 6-hour exposure to insulin, there was a downregulation in the response to insulin. Dose response studies demonstrated that 2-deoxy glucose transport was increased by 336% at 50 nmol/L insulin (P < 0.01), and reached a maximal effect at 100 nmol/L insulin (P < 0.01). Oleate and palmitate treatment did not influence basal glucose transport (without insulin stimulation), whereas insulin-stimulated glucose transport was inhibited after overnight oleate and palmitate treatment in preadipocytes and adipocytes. In 3T3-L1 preadipocytes, insulin resistance could be achieved at 0.125 mmol/L oleate or palmitate (P < 0.05, respectively), and the inhibition was dose dependent. In adipocytes, the inhibition was noted at 0.5 mmol/L oleate or 1.0 mmol/L palmitate. Visfatin mRNA expression increased during differentiation more than 1.5-fold. Bovine serum albumin (BSA) did not influence visfatin mRNA expression compared with the control group. Dose-response studies demonstrated that addition of 0.125 mmol/L oleate and palmitate to 3T3-L1 adipocytes decreased visfatin mRNA expression significantly (78%, 77%, respectively, relative to untreated control, P < 0.05), and further to 65% (relative to untreated control, P < 0.05) and 55% (relative to untreated control, P < 0.01) at 1.0 mmol/L FFA. Furthermore, the suppression on preadipocytes was similar to that of adipocytes, which reached a maximal reduction of 44% (oleate, P < 0.05) and 47% (palmitate, P < 0.05) at 1.0 mmol/L FFA.

CONCLUSIONSOleic acid and palmitic acid may induce insulin resistance in 3T3-L1 adipocytes and preadipocytes. Downregulation of visfatin mRNA may contribute to impair insulin sensitivity caused by oleate and palmitate.

3T3-L1 Cells ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Differentiation ; Cytokines ; genetics ; Dose-Response Relationship, Drug ; Gene Expression Regulation ; drug effects ; Insulin Resistance ; Mice ; Nicotinamide Phosphoribosyltransferase ; Oleic Acid ; pharmacology ; Palmitic Acid ; pharmacology ; RNA, Messenger ; analysis ; Stem Cells ; metabolism

Dizziness,chest discomfort,chest depression and dyspnea are a group of symptoms that are common complaints in clinical practice. Patients with these symptoms are usually informed that while neurosis consequent to coronary heart disease is excluded nonetheless they remain unhealthy with no rational explanation or treatment. 165 cases of these symptoms and 85 control subjects were reviewed and underwent further medical history inquiry,routine EKG test and cardiac ultrasound examination. Thirty-five patients received coronary artery angiography to exclude coronary heart disease. Serum myocardial autoantibodies against beta1-adrenoceptor,alpha-myosin heavy chain,M2-muscarinie receptor and adenine-nucleotide translocator were tested,and inflammatory cytokines and high sensitivity C-reaction protein were measured and lymphocyte subclass was assayed by flow cytometry. All patients had a complex of four symptoms or tetralogy: (1) persistent throat or upper respiratory tract infection,(2) neck pain,(3) chest pain and (4) chest depression or dyspnea,some of them with anxiety. Anti-myocardial autoantibodies (AMCAs) were present in all patients vs. 8% in and CD4-CD8+ lymphocytes were significantly higher and CD56+ lymphocytes lower in patients than those in controls (P<0.01). The ratio of serum pathogen antibodies positive against Coxsackie virus-B,cytomegalovirus,Mycoplasma pneumoniae and Chlamydia pneumoniae were all markedly higher in patients. These data led to identification of a persistent respiratory infection-related clinical syndrome,including persistent throat infection,neck spinal lesion,fib cartilage inflammation,symptoms of car-diac depression and dyspnea with or without anxiety.