Effects of fatty acid regulation on visfatin gene expression in adipocytes

Currently, adipose tissue is no longer considered as an inert tissue mainly devoted to energy storage but is emerging as an active endocrine organ. It releases a variety of factors, so-called adipocytokines, i.e. leptin,¹ resistin,¹ adiponectin,¹ acylation stimulating protein,² visfatin,³ and others. These proteins are involved in glucose and fat metabolism, and hence can influence insulin resistance. Overloading of the adipose tissue beyond its storage capacity leads to numerous free fatty acid (FFA) influxes and adipocytokine dysfunction.⁴ Moreover, the excess fat is directed towards non-adipose tissues, namely the skeletal and cardiac muscle, liver, and pancreas. In these organs, lipid accumulation can be toxic and induce insulin resistance, a phenomenon known as lipotoxicity, which further contributes to metabolic syndrome.⁵

The expansion of adipose tissue mass results from increased number and size of adipocytes.⁶ Adipocyte number is determined by recruitment of preadipocytes to adipocytes as well as cell death. The differentiation process of preadipocytes into mature adipocytes presumably occurs throughout the human life span.⁷　In addition, subcutaneous abdominal preadipocyte differentiation in vitro is inversely correlated with central obesity.⁷ Both blunted differentiation potential of preadipocytes and hypertrophic adipocytes are associated with insulin resistance. However, the difference between preadipocytes and adipocytes in response to insulin under high FFA conditions is not fully clear.

Visfatin has been identified as a new adipocytokine that directly binds to and stimulates the insulin receptor, exerting insulin-mimetic affects in vitro and in vivo.³ Moreover, visfatin is mainly secreted by visceral adipose tissues.³Elevated plasma visfatin concentrations in morbidly obese subjects are reduced after weight loss.⁸ However, it has not been elucidated how visfatin modulates glucose and lipid metabolism. According to previous data, we propose that there might be crosstalk between FFA and visfatin, which modulates insulin sensitivity to a certain degree.

Oleate (C18:1) and palmitate (C16:0) are the most prevalent monounsaturated and saturated FFA, respectively, in the circulation.⁹ The present study was designed to examine the effect of oleate and palmitate on specific glucose transport and visfatin expression in 3T3-L1 adipocytes as well as preadipocytes in vitro, which may provide some clue to the crosstalk between visfatin and insulin resistance induced by FFA.

METHODS

Materials
3T3-L1 cells were purchased from ATCC, USA. 1-methyl-3-isobutylmethylxanthine (IBMX), dexame- thasone (DEX), insulin (INS), oleic acid, palmitic acid and BSA were purchased from Sigma, USA. ³H-2-deoxy-D-glucose (2-DG) was purchased from ICN Biochemicals, Canada. TRIzol reagent was purchased from MRC, USA. The primers were synthesized by AuGCT Biotechnology,China. RNA PCR kit (AMV) was purchased from TaKaRa Biotechnology, Japan.

3T3-L1 cell culture and differentiation
3T3-L1 cells were grown in DMEM/F12 media supplemented with 10% fetal bovine serum (FBS) in 5% CO₂ at 37˚C. The cells were differentiated into adipocytes by standard procedures.¹⁰ Briefly, two-day post-confluent cells (day 0) were supplemented with 0.5 mmol/L IBMX + 1.0 µmol/L DEX + 10 µg/ml INS + 10% FBS for two days. Then, the cells were kept for two more days in culture medium with 10 µg/ml INS + 10% FBS and four to five days in culture medium with 10% FBS. The cells used for experimentation were over 80% differentiated (as determined visually). 3T3-L1 preadipocytes and adipocytes were divided into seven groups respectively for different treatments: control group (FFA-free DMEM/F12), oleate 0.125 mmol/L group, oleate 0.5 mmol/L group, oleate 1.0 mmol/L group, palmitate 0.125 mmol/L group, palmitate 0.5 mmol/L group and palmitate 1.0 mmol/L group. For glucose transport, each group included basal glucose uptake (no insulin stimulation) and insulin-induced glucose uptake (100 nmol/L insulin) and all values were derived from two to three separate experiments, performed each in triplicate. For visfatin gene expression analysis, data were representative of four to five independent experiments.

Glucose transport
Analysis of insulin on 2-deoxyglucose transport
2-deoxy-glucose (2-DG) transport in adipocytes was performed as previously described.¹¹ In brief, 3T3-L1 cells were grown as described above in 24-well plates to 70% confluence. Preadipocytes were incubated with insulin at specific concentrations (0 nmol/L－200 nmol/L) for 1 hour (or the indicated times: 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours and 6 hours ) at 37 ˚C. After one hour (or at the indicated times), glucose transport was assessed by measuring the cellular uptake of ³H-2-DG. The medium was removed and the cells were washed once with 1 ml PBS per well at 37˚C. Then, 400 µl PBS containing 50 µmol/L ³H-2-DG (specific activity 50 dpm/pmol) was added to each well and incubated at 37˚C in a water bath for 10 minutes. In all experiments, zero-time controls were used to account for background binding of ³H-2-DG (add 400 µl specific activity 50 dpm/pmol ³H-2-DG to two wells and remove the radioactive 2-DG immediately and quickly). At 10 minutes, the radioactive solution was aspirated and the cells were washed twice with 1ml of ice-cold PBS. Finally, the cells were dissolved in 400 µl of 0.1 mol/L NaOH. Aliquots of 200 µl were then transferred into a scintillation vial containing 3 ml scintillation fluid and counted with a scintillation counter. Protein concentration was determined by the method of Bradford.

FFA treatment and glucose transport
Adipocytes and preadipocytes were incubated with oleate-BSA and palmitate-BSA (molar ratio 5:1) overnight (16－18 hours). The final concentrations were 0 mmol/L, 0.125 mmol/L, 0.5 mmol/L and 1.0 mmol/L. The cells pretreated with FFA (0 mmol－1.0 mmol/L) were stimulated with 0 nmol/L (basal glucose uptake) or 100 nmol/L insulin (insulin- induced glucose uptake) for one hour at 37˚C. Glucose transport was assayed as the afore mentioned.

RNA isolation and RT-PCR amplification
Differentiating cells at the indicated time points (0, 3, 6, 9 days) and FFA treated cells were harvested by removing the medium and adding TRIzol reagent directly to the culture dishes. Total cellular RNA was isolated according to the manufacturer's instructions, quantified by spectrophotometry by A₂₆₀/A₂₈₀=1.8－2.0. Primers included visfatin sense primer: 5'- CAG TGC CTG TGT CTG TGG TCA-3', antisense primer: 5'- CTA ATG AGG TGC CAC GTC CTG-3', length: 665bp; and β-actin sense primer: 5'-ATG GGT CAG AAG GAC TCC TAT G-3', antisense primer: 5'-ATC TCC TGC TCG AAG TCT AGA G-3', length: 542 bp. The volume of reaction mixture for RT was 20 µl and for PCR was 25 µl. For RT-PCR, 1 µg RNA was reverse-transcribed with standard reagents. The cDNA was amplified by PCR under the following protocol: 2 minutes at 94˚C, 30 seconds at 94˚C, 1 minute at 60˚C, 1 minute at 72˚C for 35 cycles, followed by 7-minute extension at 72˚C. After PCR amplification, aliquots of PCR products were separated by 2.0% agarose gel electrophoresis, with the standard DNA markers scanned and analyzed by densitometry.

Statistical analysis
The results were expressed as means±standard deviation (SD). For comparison of the differences among the groups, one-way ANOVA was used and followed by a two-tailed paired, Student's t test between the control group and other groups. A P value less than 0.05 was considered statistically significant.

RESULTS

Morphological changes in 3T3-L1 cells
3T3-L1 preadipocytes were fibroblastic, and fat droplets were not present in the cytoplasm. After differentiation, bigger and rounder adipocytes were seen with multiple fat droplets in the cells, forming a “ring structure”.

Insulin resistance caused by FFA
Time- and concentration-dependent effect of insulin on glucose transport
In order to determine the response of preadipocytes to insulin, glucose transport stimulation was assessed after exposure for different times to a high concentration of insulin (100 nmol/L). The insulin effect on glucose uptake was observed by 15 minutes (439.9±230.1 zero-time group vs 1392±789.9, pmol/10 min·mg cell protein, t=3.802, P<0.05) and reached its maximum at 1 hour [(1553±836.1) pmol/10 min·mg cell protein, t=4.238, P<0.01] as compared to the zero-time group. Subsequently, there was an apparent down-regulation of insulin response by 6 hours [(498.9±116.8) pmol/10 min·mg cell protein, t=0.4282, P>0.05], at which the glucose transport was not significantly different from the zero-time value. We subsequently examined the effect of various concentrations of insulin on glucose transport. 3T3-L1 preadipocytes were exposed for 1 hour to increasing concentrations of insulin, and the effect of glucose transport was tested. Glucose transport was significantly up-regulated at a concen- tration as low as 50 nmol/L (188.5±91.5 zero-insulin vs 822.3±135.9, pmol/10 min·mg cell protein, t=19.51, P<0.01), reaching a maximal level of 100 nmol/L insulin (931.6±113.3, pmol/10 min·mg cell protein, t=16.86, P<0.01) and keeping at a stable high glucose transport rate of 200 nmol/L insulin (923.2±356.5, pmol/10 min·mg cell protein, t=5.179, P<0.01) as compared to that of 0 nmol/L insulin.

Oleic acid inducing insulin resistance in adipocytes and preadipocytes
3T3-L1 adipocytes and preadipocytes were exposed to various concentrations (0.125 mmol/L, 0.5 mmol/L and 1.0 mmol/L) of oleic acid and FFA-free DMEM/F12 overnight. Oleate treatment did not influence basal glucose transport (without insulin stimulation) compared with the control group (0 mmol/L oleate). In contrast, insulin-induced glucose uptake was impaired after oleate treatment compared with the control group (0 mmol/L oleate). The rates of glucose uptake were decreased by 3.78% (t=0.2935, P=0.7809), 36.14% (t=2.070, P<0.05), and 48.19% (t=2.800, P=0.038) in adipocytes and by 22.14% (t=2.598, P<0.05), 29.26% (t=4.922, P<0.01), and 48.91% (t=3.605, P<0.01) in preadipocytes, respectively.

Palmitic acid inducing insulin resistance in adipocytes and preadipocytes
3T3-L1 adipocytes and preadipocytes were exposed to various concentrations of palmitate (0.125 mmol/L, 0.5 mmol/L and 1.0 mmol/L) and FFA-free DMEM/F12 for 16－18 hours. The rates of insulin-stimulated glucose uptake were decreased by 10.62% (t=0.4605, P>0.05), 19.40% (t=1.684, P>0.05), and 43.41% (t=3.876, P<0.05) in adipocytes and by 41.84% (t=3.256, P<0.05), 49.03% (t=3.537, P<0.01), and 64.96% (t=6.794, P<0.01) in preadipocytes as compared to those of the control group (FFA-free DMEM/F12). However, there was no difference in basal glucose transport (without insulin stimulation) after palmitate treatment.

Increase of visfatin mRNA expression during the differentiation of 3T3-L1 cells
Visfatin mRNA was expressed in 3T3-L1 preadipocytes (day 0) at low levels and increased significantly by 1.5-fold on day 6 compared with day 0 (t=3.367, P<0.05). Then visfatin mRNA expression remained at a high level to day 9 (t=3.209, P<0.05, Fig. 1).

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Fig. 1. Expression of visfatin in 3T3-L1 cells during differentiation. A: Confluent preadipocytes (day 0) were differentiated and at the indicated days (0, 3, 6, 9) total RNA was subjected to RT-PCR. Visfatin mRNA are shown relative to cells on day 0 (100%) for an average of four experiments. *P<0.05 vs confluent cells (day 0). B: Lane 1: 0 d; Lane 2: 3 d; Lane 3: 6 d; Lane 4: 9 d; Lane 5: 100-bp DNA ladder. Expression of visfatin in 3T3-L1 cells during differentiation.

Oleic acid decreasing visfatin mRNA in 3T3-L1 adipocytes and preadipocytes
The addition of oleic acid and incubating for 16－18 hours dose-dependently suppressed visfatin mRNA, with a 22% reduction at concentration as low as 0.125 mmol/L (t=3.270, P<0.05) and a maximal 35% reduction at 1.0 mmol/L (t=2.922, P<0.05) in 3T3-L1 adipocytes (Figs. 2A and 3A). A similar effect was seen in preadipocytes, with a significant 31% suppression at 0.5 mmol/L (t=3.405, P<0.05) and a maximal 44％ suppression at 1.0 mmol/L (t=5.434, P<0.05) (Figs. 2B and 3B).

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Fig. 2. Dose-dependent regulation of visfatin mRNA by oleate in 3T3-L1 adipocytes (A) and preadipocytes (B). Various concentrations of oleate were added for 16h－18h. Total RNA was extracted and subjected to RT-PCR to determine visfatin mRNA levels normalized to β-actin as described in materials and methods. Results are expressed as mean±SD of four to five separate experiments. *P<0.05, **P<0.01 vs 0 mmol/L oleic acid treated cells.

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Fig. 3. Effect of oleate on visfatin mRNA in 3T3-L1 adipocytes (A) and preadipocytes (B). Lane 1: control; Lane 2: 0.125mmol/L oleate; Lane 3: 0.5mmol/L oleate; Lane 4: 1.0mmol/L oleate; Lane 5: 100-bp DNA ladder.

Palmitic acid downregulating visfatin expression in 3T3-L1 adipocytes and preadipocytes
We determined dose-dependent effects of palmitic acid on visfatin mRNA. A negative effect on visfatin was seen at concentrations of 0.125 mmol/L (t=3.410, P<0.05) and 0.5 mmol/L (t=4.760, P<0.05) in 3T3-L1 adipocytes and preadipocytes respectively (Figs. 4 and 5). Palmitic acid maximally downregulated visfatin expression by 45% (t=5.299, P<0.01) and 47% (t=3.232, P<0.05) at 1.0mmol/L in both cell types. No difference was detected between the control and BSA groups (P>0.05).

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Fig. 4. Dose-dependent regulation of visfatin mRNA by palmitate in 3T3-L1 adipocytes (A) and preadipocytes (B). Various concentrations of palmitate were added for 16h－18h. Total RNA was extracted and subjected to RT-PCR to determine visfatin mRNA levels normalized to β-actin as described in materials and methods. Results are expressed as the mean±SD of four to five separate experiments. *P<0.05, **P<0.01 vs 0 mmol/L palmitic acid treated cells.

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Fig. 5. Effect of palmitate on visfatin mRNA in 3T3-L1 adipocytes (A) and preadipocytes (B). Lane 1: 100 bp DNA ladder; Lane 2: control; Lane 3: 0.2 mmol/L BSA; Lane 4: 0.125 mmol/L palmitate; Lane 5: 0.5 mmol/L palmitate; Lane 6: 1.0 mmol/L palmitate.

DISCUSSION

Recent data from the USA,¹² Australia,¹³ China¹⁴ and a number of European countries^15,16 consistently demonstrate that the prevalence of overweight and obesity is increasing exponentially in all age groups. Obesity is a global public health concern. At a workshop held in April 2005, the International Diabetes Federation (IDF) issued a new global definition of metabolic syndrome which puts the emphasis on central obesity as the essential component.¹⁷ It is well-established that obesity, particularly visceral obesity, is a major contributor to chronic disease and disability, such as dyslipidemia, hypertension, type 2 diabetes and cardiovascular disease.¹⁸ A major link between visceral obesity and metabolic complications is excessive adipose tissue lipolysis and the consequent high plasma FFA.¹⁹ Several studies have examined the short-term and long-term effects of fatty acids on glucose transport in adipocytes.^20-22 In the current study, both monounsaturated FFA oleate and saturated FFA palmitate inhibited insulin stimulation of glucose transport directly after overnight addition of fatty acids to 3T3-L1 preadipocytes and adipocytes. In addition, the suppression was more severe in preadipocytes than in adipocytes. One way to explain this observation is that both FFA esterification and the esterification /oxidation ratio in preadipocytes were much lower than in adipocytes.²³ Nonetheless, both present and previous studies^20-22 indicate that fatty acids can impair insulin-mediated glucose transport in adipocytes and preadipocytes. These findings imply that fatty acids may induce insulin resistance directly.

At present, a number of adipocytokines have also been implicated in the induction of insulin resistance in obesity.²⁴ The recently discovered adipocytokine visfatin preferentially produced in visceral adipose tissue, can be found in skeletal muscle, liver, bone marrow and lymphocytes, where it was initially identified as preB-cell colony-enhancing factor (PBEF).²⁵ Recently, Fukuhara et al³ demonstrated that visfatin had insulin-mimetic effects. As does insulin, visfatin increases lipogenesis and enhanced glucose uptake in 3T3-L1 adipocytes and L6 myocytes. In addition, visfatin suppresses glucose release in hepatocytes. More interesting, when delivered directly to diabetic mice, visfatin also improves insulin sensitivity, while decreasing the levels of glucose and insulin in vivo. Although the affinity of visfatin for the insulin receptor appears to be similar to that of insulin, there are important differences between visfatin and insulin. The level of plasma visfatin is much lower (3%－10% of the insulin concentration) under physiological conditions, and it is also not regulated by fasting and feeding. These changes may be attributable to its small contribution to plasma glucose level under physiological conditions.³ In contrast, the levels of visfatin in plasma and visceral adipose tissue of the obese and type 2 diabetics increase dramatically. Furthermore, plasma visfatin concentrations correlated strongly with the amount of visceral fat.^3,8,26 These data show that visfatin may play an important role in the pathophysiology of obesity and obesity-related disorders. Most obese patients are known to present high concentrations of plasma FFA, which would lead to decreased glucose uptake in insulin-sensitive tissues, especially adipose tissues. This in turn leads to a tissue-specific disorder in insulin response, increased level of plasma insulin, and lipotoxicity.⁴

In our study, we found for the first time that oleate and palmitate significantly downregulated visfatin mRNA in 3T3-L1 adipocytes as well as preadipocytes. The suppressive effects were enhanced as the dose of FFA increased, reaching a maximum of 44% (for oleate) and 47% (for palmitate). But the result is not inconsistent with that of previous studies. It is apparent that a significantly large number of macrophages are infiltrated into the expanding adipose tissue in obese subjects.²⁷ Visfatin was predominantly produced and released by macrophages of the visceral white adipose tissue in obesity, but not adipocytes.²⁸ Macrophage infiltration may account for the high visfatin mRNA expression in visceral white adipose tissues. The proinflammatory factors like FFA, such as interleukin-6 and tumor necrosis factor-α are increased in obesity.²⁷ They both suppressed visfatin mRNA expression in adipocytes.^29,30 Accordingly, we conclude that downregulation of visfatin in 3T3-L1 adipocytes and preadipocytes may contribute to fatty acid induced glucose intolerance under conditions of lipid overload.

Visfatin is a newly identified adipocytokine that is highly secreted by visceral fat, especially by macrophages, and increases with differentiation of preadipocytes. It remains to be established whether visfatin production is a compensatory response to the imbalance in glucose and lipid metabolism in tissue-specific insulin resistance or a triggered factor to impair glucose tolerance.

Taken together, oleate and palmitate can impair insulin-stimulated glucose uptake in a dose- dependent manner in 3T3-L1 adipocytes and preadipocytes. Downregulation of visfatin mRNA may contribute to impaired insulin sensitivity caused by oleate and palmitate in vitro.

REFERENCES

1． Koerner A, Kratzsch J, Kiess W. Adipocytokines: leptin--the classical, resistin--the controversial, adiponectin--the promising, and more to come. Best Pract Res Clin Endocrinol Metab 2005; 19: 525-546.

2． Maslowska M, Wang HW, Cianflone K. Novel roles for acylation stimulating protein/C3adesArg: a review of recent in vitro and in vivo evidence. Vitam Horm 2005; 70: 309-332.

3． Fukuhara A, Matsuda M, Nishizawa M, Segawa K, Tanaka M, Kishimoto K, et al. Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science 2005; 307: 426-430.

4． Faraj M, Lu HL, Cianflone K. Diabetes, lipids, and adipocyte secretagogues. Biochem Cell Biol 2004; 82: 170-190.

5． Slawik M, Vidal-Puig AJ. Lipotoxicity, overnutrition and energy metabolism in aging. Aging Res Rev 2006; 5: 144-164.

6． Bakker AH, Van Dielen FM, Greve JW, Adam JA, Buurman WA. Preadipocyte number in omental and subcutaneous adipose tissue of obese individuals. Obes Res 2004; 12: 488-498.

7． Permana PA, Nair S, Lee YH, Luczy-Bachman G, Vozarova De Courten B, Tataranni PA. Subcutaneous abdominal preadipocyte differentiation in vitro inversely correlates with central obesity. Am J Physiol Endocrinol Metab 2004; 286: E958-E962.

8． Haider DG, Schindler K, Schaller G, Prager G, Wolzt M, Ludvik B. Increased plasma visfatin concentrations in morbidly obese subjects are reduced after gastric banding. J Clin Endocrinol Metab 2006; 91: 1578-1581.

9． Gaster M, Rustan AC, Beck-Nielsen H. Differential utilization of saturated Palmitate and unsaturated oleate: evidence from cultured myotubes. Diabetes 2005; 54: 648-656.

10． Chae GN, Kwak SJ. NF-κB is involved in the TNF-α induced inhibition of the differentiation of 3T3-L1 cells by reducing PPARγ expression. Exp Mol Med 2003; 35: 431-437.

11． Maslowska M, Sniderman AD, Germinario R, Cianflone K. ASP stimulates glucose transport in cultured human adipocytes. Int J Obes 1997; 21: 261-266.

12． Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of overweight and obesity in the United States, 1999-2004. JAMA 2006; 295: 1549-1555.

13． Booth ML, Chey T, Wake M, Norton K, Hesketh K, Dollman J, et al. Change in the prevalence of overweight and obesity among young Australians, 1969–1997. Am J Clin Nutr 2003; 77: 29-36.

14． Wang Y, Mi J, Shan XY, Wang QJ, Ge KY. Is China facing an obesity epidemic and the consequences? The trends in obesity and chronic disease in China. Int J Obes (Lond) 2006 [Epub ahead of print].

15． Lobstein TJ, James WP, Cole TJ. Increasing levels of excess weight among children in England. Int J Obes Relat Metab Disord 2003; 27: 1136-1138.

16． Rennie KL, Jebb SA. Prevalence of obesity in Great Britain. Obes Rev 2005; 6: 11-12.

17． Reaven GM. The metabolic syndrome: requiescat in Pace. Clin Chem 2005; 51: 931-938.

18． Poirier P, Giles TD, Bray GA, Hong Y, Stern JS, Pi-Sunyer FX, et al. Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight Loss: an update of the 1997 American Heart Association Scientific Statement on obesity and heart disease from the obesity committee of the council on nutrition, physical activity, and metabolism. Circulation 2006; 113: 898-918.

19． Koutsari C, Jensen MD. Free fatty acid metabolism in human obesity. J Lipid Res 2006; 47: 1643-1650.

20． Van Epps-Fung M, Williford J, Wells A, Hardy RW. Fatty acid-induced insulin resistance in adipocytes. Endocrinology 1997; 138: 4338-4345.

21． Chavez JA, Summers SA. Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 2003; 419: 101-109.

22． Hunnicutt JW, Hardy RW, Williford J, McDonald JM. Saturated fatty acid-induced insulin resistance in rat adipocytes. Diabetes1994; 43: 540-545.

23． Carnicero HH. Changes in the Metabolism of long chain fatty acids during adipose differentiation of 3T3 L1 Cells. J Biol Chem 1984; 259: 3844-3850.

24． Fantuzzi G. Adipose tissue, adipokines and inflammation. J Allergy Clin Immunol 2005; 115: 911-919.

25． Sethi JK, Vidal-Puig A. Visfatin: the missing link between intra-abdominal obesity and diabetes? Trends Mol Med 2005; 11: 344-347.

26． Chen MP, Chung FM, Chang DM, Tsai JC, Huang HF, Shin SJ, et al. Elevated plasma level of visfatin/pre-B cell colony-enhancing factor in patients with type 2 diabetes mellitus. J Clin Endocrinol Metab 2006; 91: 295-299.

27． Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa KI, Kitazawa R, et al. MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest 2006; 116: 1494-1505..

28． Curat CA, Wegner V, Sengenes C, Miranville A, Tonus C, Busse R, et al. Macrophages in human visceral adipose tissue: increased accumulation in obesity and a source of resistin and visfatin. Diabetologia 2006; 49: 744-747.

29． Kralisch S, Klein J, Lossner U, Bluher M, Paschke R, Stumvoll M, et al. Hormonal regulation of the novel adipocytokine visfain in 3T3-L1 adipocytes. J Endocrinol 2005; 185: R1-R8.

30． Kralisch S, Klein J, Lossner U, Bluher M, Paschke R, Stumvoll M, et al. Interleukin-6 is a negative regulator of visfatin gene expression in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 2005; 289: E586-E590.