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Visfatin Is an Adipokine, But It Is Not Regulated by Thiazolidinediones

Lundberg Laboratory for Diabetes Research (A.H., V.R.S., S.G., P.-A.J., U.S.), Department of Internal Medicine, Sahlgrenska Academy, Goteborg University, Goteborg, Sweden; and Department of Medicine (J.P., M.L.), University of Kuopio, SE-413 45 Kuopio, Finland

Address all correspondence and requests for reprints to: Dr. Ann Hammarstedt, Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, Sahlgrenska University Hospital, SE-413 45 Goteborg, Sweden. E-mail: ann.hammarstedt{at}medic.gu.se.

	Abstract

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Context: Visfatin was recently reported to be expressed in human adipose tissue and to exert insulin-mimicking effects.

Objective: The objective of this study was to examine whether visfatin is a true adipokine and is expressed in isolated fat cells. We also examined whether visfatin is regulated by thiazolidinediones and, thus, can contribute to the ability of these agents to improve insulin sensitivity.

Design: This was an open-labeled drug therapy trial.

Setting: This study was performed at a university hospital.

Patients: Seven newly diagnosed and previously untreated type 2 diabetic patients and six healthy individuals with reduced insulin sensitivity participated in the study.

Intervention: Pioglitazone therapy (30–45 mg/d) was given for 3–4 wk.

Main Outcome Measures: Serum and adipose tissue mRNA levels of visfatin and adiponectin were the main outcome measures.

Results: Visfatin mRNA is expressed in both adipose tissue and isolated adipocytes. Treatment with thiazolidinediones for 3–4 wk did not alter the gene expression or circulating levels of visfatin in either nondiabetic or the diabetic individuals, whereas adiponectin increased significantly.

Conclusion: The present study shows that visfatin is a true adipokine, but it is not regulated by TZD and, thus, is unlikely to contribute to the insulin-sensitizing actions of these drugs.

	Introduction

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VISFATIN WAS RECENTLY reported to be expressed in human adipose tissue, in particular in visceral fat (1). The cDNA sequence is identical with pre-B cell colony-enhancing factor, a growth factor for early B cells also previously shown to be a secreted protein expressed in bone marrow, liver, muscle, and human fetal membranes (2, 3).

Visfatin was found to exert insulin-mimicking effects, such as lowering plasma glucose levels after adenoviral-mediated expression in vivo in mice (1). Additionally, studies addressing the molecular mechanisms revealed that visfatin activates the intracellular signaling cascade for insulin, including tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1/2 (IRS1/2) as well as downstream activation of protein kinase B/Akt. Interestingly, however, visfatin activates the insulin receptor in a manner distinct from that of insulin (1). Characterizing this activation step will be an important challenge, because this may identify novel targets for the development of antidiabetic therapy.

Because visfatin was identified from adipose tissue rather than primary human fat cells, it is not clear whether this molecule is a true adipokine, i.e. truly expressed in the fat cells rather than the stromovascular cells, which, in turn, have been shown to secrete several cytokines and peptides (4, 5, 6).

An additional and interesting question is whether visfatin expression in human adipose cells and/or adipose tissue is regulated by the thiazolidinediones (TZD) and, thus, can contribute to the ability of these agents to improve insulin sensitivity.

We, therefore, examined both the circulating levels of visfatin as well as its mRNA expression in isolated fat cells and intact adipose tissue from five insulin-resistant, but nondiabetic, individuals before and after 3 wk of treatment with 30 mg pioglitazone daily. These individuals represent a subgroup from our recent study in which we examined the effects of TZD on gene expression and insulin signaling in adipose cells and tissue (7). Additionally, we studied circulating visfatin levels and mRNA expression in the abdominal sc adipose tissue of seven newly diagnosed and untreated type 2 diabetic individuals before and after 4 wk of therapy with 45 mg pioglitazone daily.

	Subjects and Methods

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Subjects

This study was approved by the ethical committees of Goteborg University and University of Kuopio, and informed consent was obtained from each subject.

Nondiabetic subjects

Ten male nondiabetic subjects, previously identified as having low expression of IRS-1 and glucose transporter-4 protein in adipose cells, which in several studies we have shown to be markers of insulin resistance (8, 9), volunteered to undergo a 3-wk treatment period with pioglitazone (Actos; 30 mg/d; Takeda Pharmaceuticals, Chicago, IL). These individuals have been described in detail previously (7). Fasting serum and RNA samples from the abdominal sc adipose tissue as well as isolated adipose cells were available from six and five subjects, respectively, for the present analyses. The characteristics of these subjects before and after TZD treatment are shown in Table 1.

Seven individuals (five women and two men) with newly detected and previously untreated type 2 diabetes were recruited. Fasting serum samples and biopsies from the abdominal sc adipose tissue were obtained before and after 4 wk of treatment with pioglitazone (Actos; 45 mg/d). The characteristics of these individuals before and after TZD treatment are shown in Table 2. Intraabdominal (visceral) fat was measured by a computed tomography (CT) scan.

Insulin sensitivity was measured with the euglycemic, hyperinsulinemic clamp technique, essentially as described previously (7). In brief, insulin was infused at a constant rate of 40 mU/m²·min for the nondiabetic subjects and at 80 mU/m²·min for the diabetic subjects. The blood glucose level was maintained at 5 mmol/liter. Glucose was analyzed in venous blood using an automatic glucose analyzer (YSI Instrument Co., Yellow Springs, OH), and insulin was measured with a standard RIA.

Biochemical analyses

Circulating adiponectin levels were measured in plasma by an ELISA [B-Bridge International, Sunnyvale, CA; coefficient of variation (CV), 2.3%], and visfatin (pre-B cell colony-enhancing factor) was determined by an enzyme immunoassay (Phoenix Pharmaceuticals, Belmont, CA; CV, 13.0%).

Fat cell isolation was performed as previously reported (7). Briefly, biopsies were washed to remove traces of blood and were treated with 0.8 mg/ml collagenase (Sigma-Aldrich Corp., St. Louis, MO) for approximately 60 min at 37 C. Isolated adipose cells were filtered through a 250-µm pore size nylon mesh and washed four times with fresh medium to remove collagenase, and cell size was measured.

mRNA isolation and quantification

Total cellular RNA was extracted using the guanidinium thiocyanate method, and TaqMan real-time RT-PCR was used for quantification of mRNA levels essentially as previously described (7, 8, 9). The sequences for the primers and probes are available on request. The quantity of a particular gene in each sample was normalized to that of 18S rRNA (CV, 5.7%).

Statistical analysis

Conventional statistical methods were used (Stat View, SAS Institute, Inc., Cary, NC). Differences were tested with nonparametric Wilcoxon’s test for paired observations. Correlations were analyzed with Spearman’s nonparametric test.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Table 1 shows the clinical characteristics of the nondiabetic subjects as well as the results of the circulating adiponectin and visfatin levels before and after 3 wk of treatment with pioglitazone. Both the insulin-stimulated glucose disposal rate during the clamp and the adiponectin mRNA and circulating levels were increased. However, both adipose cell and adipose tissue mRNA as well as the circulating levels of visfatin were unchanged. Importantly, visfatin expression was clearly detected in both adipose tissue and isolated adipose cells. Thus, visfatin appears to be a true adipokine.

Similar results were obtained in the newly diagnosed type 2 diabetic individuals (Table 2). Both fasting glucose and insulin levels were significantly reduced after 4 wk of treatment with pioglitazone, and the glucose disposal rate was also increased. Circulating and mRNA adiponectin levels were markedly increased after 4 wk of treatment. Again, however, circulating visfatin levels as well as mRNA expression in adipose tissue remained unchanged.

We also examined whether circulating visfatin levels correlated with adiponectin levels either before or after treatment with pioglitazone, but no correlations were found (data not shown). Interestingly, circulating visfatin levels were approximately 2-fold higher in the type 2 diabetic than in the nondiabetic individuals (Table 2). Similarly, visfatin mRNA levels were considerably (7- to 8-fold) higher in the adipose tissue of the type 2 diabetic compared with the nondiabetic individuals. This is, at least in part, due to the differences in body weight, because a positive, albeit weak, correlation was found between body mass index and circulating visfatin levels (data not shown). In addition, potential differences related to the gender distribution cannot be excluded. No significant correlation was seen between circulating or mRNA levels of visfatin and intraabdominal fat content, measured by CT scan, in the diabetic group (data not shown). Because we did not measure intraabdominal fat in the nondiabetic group, we used the waist/hip ratio (WHR) and waist circumference as surrogate markers. Again, no correlation was seen with circulating levels of visfatin. To increase the statistical power, we pooled the data from both groups. Again, however, no significant correlation was seen between WHR or waist circumference and visfatin levels (data not shown).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In the present study we studied whether visfatin, identical with pre-ß cell colony-enhancing factors (3), is expressed in isolated fat cells as well as intact adipose tissue and whether the expression is regulated by the insulin-sensitizing TZDs. Visfatin was clearly expressed in the isolated sc adipose cells and, thus, can be considered a true adipokine. The mRNA levels were also higher in the isolated adipose cells than in the adipose tissue biopsies, which also supports this concept.

Because the visfatin gene does not contain a clear signal sequence for secretion, studies of the circulating visfatin levels before and after TZD are also important. However, treatment with TZD for 3–4 wk did not increase mRNA or circulating visfatin levels in either group. These data clearly suggest that visfatin is not a target for TZD and, thus, is unrelated to the insulin-sensitizing effect of these drugs.

Interestingly, both circulating levels and mRNA expression of visfatin were increased in the diabetic vs. nondiabetic individuals. One likely reason for this is the different body weights of the two groups. A correlation, albeit weak, was found between body mass index and circulating visfatin levels. Unfortunately, we did not characterize the amount of visceral fat in all individuals. However, using WHR or waist circumference as a surrogate marker for visceral fat showed no significant correlation with circulating levels of visfatin. We also did not find any correlation between the amount of visceral fat, measured with a CT scan, and visfatin levels in the diabetic group. Visfatin was reported to be more highly expressed in visceral than sc fat (1), and visceral fat may be increased in insulin-resistant individuals (10). However, similar to our results, a recent extensive study in humans found no difference in visfatin mRNA levels between visceral and sc adipose tissue (10).

A recent experimental study examined the effect of IL-6 on visfatin gene expression in 3T3-L1 adipose cells (11). It was reported that this cytokine is a negative regulator of visfatin expression, and interestingly, addition of TZD did not overcome this inhibitory effect (11). Thus, TZDs do not appear to increase visfatin expression in either human adipose cells in vivo or in 3T3-L1 cells in vitro.

In conclusion, the present study shows that visfatin is a true adipokine, but it is not regulated by TZD. We report that both mRNA and circulating levels of visfatin are increased in obese type 2 diabetic subjects, and this may be related to their increased adipose tissue mass and/or other factors related to the diabetic state.

	Footnotes

 
This work was supported by the Swedish Research Council (K2004-72X-03506-33A), the European Community (FP6 EUGENE2, LSHM-CT-2004-512013), the Swedish Diabetes Association, the Novo Nordisk Foundation, the Sonya Hedenbratt Memorial Fund, the IngaBritt and Arne Lundberg Foundation, and the Torsten and Ragnar Söderberg Foundation.

First Published Online January 4, 2006

Abbreviations: CT, Computed tomography; CV, coefficient of variation; IRS-1/2, insulin receptor substrate-1/2; TZD, thiazolidinedione; WHR, waist/hip ratio.

Received June 23, 2005.

Accepted December 21, 2005.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 91/3/1181    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hammarstedt, A. Articles by Smith, U. Search for Related Content PubMed PubMed Citation Articles by Hammarstedt, A. Articles by Smith, U. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Medline Plus Health Information Diabetes Related Collections Metabolism