Dynamics of serum visfatin level after abdominal surgery: a new proinflammatory marker in the early diagnosis?

Summary

Background. Visfatin is an adipocytokine produced primarily by visceral adipose tissue. In addition to its effect on the insulin receptor, it is a proinflammmatory cytokine whose levels may vary in the presence of inflammatory processes. The aim of our study was to correlate changes in the serum levels of visfatin in the early period after a post-abdominal surgery procedure with the serum levels of other proinflammatory cytokines.

Methods and results. Our prospective cross-sectional study included 20 patients undergoing elective laparotomic resection of the colon. The parameters determined in our study were visfatin, leptin, resistin, adiponectin, TNF α, interleukin-6 and C-reactive protein levels, with the dynamics of changes in the above markers compared assessed at +12, +24, +48, and +72 hours after the procedure.

The serum levels of visfatin peaked as early as 24 hours post-surgery returning to normal after 72 hours. TNF α and IL-6 levels reached their maximum 12 to 24 hours later while CRP levels peaked after 72 hours.

Conclusions. Significantly increased serum levels of visfatin detected in the early period after abdominal surgery preceded the increase in the levels of other proinflammatory markers, TNF α, IL-6, and CRP.

Given its dynamics, visfatin could serve as an early predictor of development of inflammatory changes in patients undergoing surgery, particularly those with visceral obesity.

Key words:
visfatin, abdominal surgery, proinflammatory cytokines, visceral obesity

Visfatin is an adipocytokine produced primarily by visceral adipose tissue and recently characterized in more detail (1). Its metabolic effects include modulation of the effect of insulin by binding to the insulin receptor at a site different from the insulin receptor. Originally, visfatin was characterized as a growth factor for B-cell proliferation, so called pre-B cell colony-enhancing factor (PBEF) in lymphocytes. Pre-B cell colony-enhancing factor was subsequently identified as a ubiquitous cytokine present in a variety of cells and tissues such as neutrophil leukocytes, hepatic, cardiac, and muscle tissue (2,3,4,5).

In a study by Fukuhara et al. (6), PBEF was referred to as a factor with an insulin-mimetic effect. Its levels correlate with those of adiponectin and have an effect on insulin resistance. In experimental studies, PBEF levels were increased in hyperglycemia and decreased after the administration of insulin and somatostatin. It is directly associated with nutrient availability and cell bioenergetics. In newer studies, visfatin has been implicated as an enzyme responsible for intra- and extracellular NAD biosynthesis. Moreover, visfatin interferes with glucose homeostasis and exerts a major hypoglycemic action modulating also B-cell function. Of interest are also the findings documenting changes in visfatin levels post-exercise: the short-term increase immediately after a bout of exercise, and the long-term decrease after regular long-term exercise are no doubt related to glucose and insulin levels. Visfatin levels may also be affected by a change in body weight, i.e., loss of adipose tissue. In addition, visfatin stimulates interleukin (IL)-6 and IL-8 expression and has been shown to be associated with oxidative stress parameters.

Visfatin is significantly contained in adipose tissue, predominantly in visceral fat and, hence,  is closely associated with visceral obesity. A relationship between obesity, serum levels of visfatin and its mRNA expression  as well as between visfatin and BMI has been conclusively documented (7). While an association with visceral obesity has been confirmed, this was not the case of subcutaneous fat. Increased circulating visfatin levels have also been documented in metabolic syndrome patients.

Adipose tissue is composed of mature adipocytes and stromal cells including macrophages, lymphocytes, endothelial cells, and preadipocytes. All these cells exhibit most active endocrine (paracrine and autocrine) activity and are involved in energetic homeostasis regulation and tissue remodeling (8,9).

Obesity is associated with changes in adipocyte production. Among the so-called classic adipocytokines, the plasma levels of leptin correlates with BMI a a other markers of body composition (10). On the other hand, adiponectin levels are decreased in women, and resistin may significantly affect insulin resistance (11).

It follows from the above that adipose tissue may be significantly involved in the metabolic and immune response of the body to stress.

Recent studies have shown that visfatin is also closely related to immune processes and is a major proinflammatory cytokines in the variety of inflammatory processes (e.g., acute inflammation of the lung or sepsis) (12,13,14). In these conditions, adiponectin has antagonistic properties. Increased levels of visfatin have also been shown to correlate with other proinflammatory cytokines such as IL-1, IL-6, and TNF α.

Visfatin has also been investigated in surgical patients. In patients with cholecystolithiasis and subsequent cholecystectomy, visfatin levels were found to be increased in the long run (1 year after surgery (15). Visfatin levels were also found to be decreased in patients with decreased BMI and adipose tissue. Following gastroplasty, visfatin levels, visfatin levels depended on  on body weight changes declining in the early postoperative period at 4 months to subsequently increase at the end of 1-year follow-up, correlating with IL-6 levels and plasma insulin concentration (16). On the other hand, no changes in visfatin levels were documented in a prospective study of 27 morbidly obese women (BMI over 40 kg/m²) on followed up for 1 year following biliopancreatic diversion despite a significant fall in BMI (from 46.1± 13.1 to 35.1± 5.7 kg/m²) (17).

A study analyzing 200,000 deaths due to sepsis in the USA showed that visfatin levels significantly rise in sepsis patients and correlates significantly with the risk ratio of death (18). These conclusions were also confirmed in experimental studies (19) demonstrating that IL-6 was was a regulator of the visfatin gene in 3T3-L1 adipocytes.

The issue of early diagnosis of inflammatory changes in the perioperative period received attention also in the Czech literature. Proinflammatory cytokine monitoring may largely affect prognosis of patients in terms of early diagnosis of postoperative intraabdominal sepsis (20). Based on the dynamics of changes in cytokines, it is possible to monitor the balance between proinflammatory cytokines and other regulatory mechanisms critical for the development of an inflammatory process, in particular in at-risk patients (21,22). Given the above facts, mention should be made of an important study of the adipocytokine leptin, which seems to be a very early marker of postoperative stress in the perioperative period (23).

Our previous study (24) was designed to monitor selected proinflammatory adipose tissue cytokines prior to and after scheduled surgery (laparoscopic cholecystectomy) in patients with various stages of reduction in renal function and obesity. Our study noted a significant increase in the levels of proinflammatory adipocytokines and  increased infiltration of immunocompetent cells in obese patients (BMI over 30 kg/m²) compared with non-obese individuals. Decreased visfatin levels were also found in non-obese kidney recipients (25).

The aim of the present study was to test the hypothesis whether and how serum visfatin levels vary in the early perioperative period depending on the possibility of developing infection (a proinflammatory marker) using dynamic temporal perioperative monitoring at 0-12-24-48, and 72 hours, and whether its dynamics differs from that of other proinflammatory cytokines already in use. We also sought to determine whether or not visfatin levels vary depending on the amount of adipose tissue in the preoperative period. All study patients had elective intraabdominal surgery (segmental resection of the colon). Scheduled abdominal laparotomy was chosen as a model of non-infective inflammatory stimulation of the cytokine cascade, which some authors have suggested may play a major role in the activation of visfatin production.

Study patients and methods

The study was conducted in conformity with the principles of the Declaration of Helsinki including the current version of current Good Clinical Practice. The selected therapeutic procedures were consistent with institutional rules. Prior to inclusion into the study, all patients received detailed information and gave their informed. The study was performed at the Department of Surgery of Charles University School of Medicine 3 between 1 January 2010 and 30 September 2012; laboratory investigations were performed at the Department of Nephrology of the Transplant Center of the Institute for Clinical and Experimental Medicine and the Institute of Rheumatology of the Charles University School of Medicine 1 and General University Hospital.

The prospective cross-sectional study included 20 patients having elective laparotomic segmental resection of the colon. Of this number, 10 patients were heavily obese (BMI over 30 kg/m², Group I). Group II was made up by the other 10 patients with a BMI below 30 kg/m². As regards associated disease, 4 Group I patients had Type-2 diabetes mellitus, present in 3 patients of Group II. Diabetes mellitus was treated using standard oral hypoglycemic agents and lifestyle modifications. Each group included 5 patients with well-controlled systolic-diastolic hypertension. Patients with surgical intraoperative and postoperative complications were not eligible. All patients had baseline anthropometric examination 1 day before the procedure and their BMI was calculated. Blood samples for biochemistry were obtained as part of the preoperative examination and subsequently at 12, 24, 48, and 72 hours upon completion of the procedure. Serum was obtained by centrifugation and samples stored first at –20ºC and next at –70ºC for further analysis.

Serum visfatin was determined by enzyme-linked immunosorbent assay (ELISA) according to the protocol of Bio Vision Research Products, Mountain View, CA, USA. Serum adiponectin was determined using commercially available ELISA kit (Bio Vendor, Brno, Czech Republic, Linco Research, St Charles, MI, USA). Serum IL-6 and TNF α levels were determined using a human serum adipokine LINCOplex kit (Luminex 200 instrument Linco Research) while serum C-reactive protein (CRP) levels were measured using the ultrasensitive CRP ELISA kit (DSL, Oxon, UK). Other standard biochemical parameters were determined using a device manufactured by Olympus Diagnostica GbmH, Hamburg, Germany. Serum insulin was determined by RIA (Cis Bio Int, Lyon, France), and glycosylated hemoglobin (HbA_1c) by liquid chromatography on a Tosohi HLC-723 G7 system (Shiba, Minato-ku, Tokyo, Japan).

Statistical analysis

Statistical analysis was performed with Sigmastat software (SPSS Inc, Chicago, IL, USA). Paired t-test and two-sample t-test were employed to compare results of both patient groups. Results were considered statistically significant if p < 0.05.

Results

Basic clinical and laboratory parameters of both patient groups prior to abdominal surgery are shown in Table 1. The groups did not differ in age and sex, serum creatinine, cholesterol and diastolic BP. Only slight, yet significant differences (p < 0.05) were observed in systolic BP, fasting glycemia, and insulin. More markedly significant differences (p < 0.02) were shown in HbA_1c and triglyceride levels. These findings may relate to the significant differences in BMI (p < 0.02) between the two groups (31.2± 4.2 vs 27.3±3.3 kg/m²).

The serum levels of selected adipocytokines (visfatin, leptin, resistin, adiponectin) and other proinflammatory cytokines (TNF α, IL-6, and CRP) in the preoperative period are shown in Table 2. No significant differences in the preoperative levels of the above cytokines were found except for adiponectin (higher values in Group II) and IL-6 (higher levels in Group I) (both p < 0.05).

Table 3 gives the mean values and standard deviations of selected adipocytokines and proinflammatory cytokines in the perioperative period (0h, +12h, +24h, + 48h, and +72 hours post-surgery) in either group. The table also shows the dynamics of changes of individual parameters relative to baseline. It is evident from the table that visfatin levels rise dynamically significantly already in the early perioperative period (from +12h onward), more in Group I, and there are significant differences between the groups (p < 0.02). In either group, peak values were reached at +24h periods (p < 0.01); however, again with differences between the groups (p < 0.01). A slow decline began to occur in the +48h period (p < 0.01), with another decrease in the +72h period. However, the significant differences between the two groups persisted (p < 0.02), with the values higher in Group I.

An  analogous trend on perioperative changes in serum levels was noted with leptin (maximum rise in +24h periods), showing significant changes in either group, with higher values in Group I. On the other hand, a reverse trend was seen in the dynamics of changes in adiponectin levels, with highest values observed in both groups preoperatively with a dynamically significant decline beginning with period +24h (p < 0.02). There were significant differences between the groups, with lowest values in Group I. Adiponectin levels correlated inversely with the proinflammatory cytokines TNF α and IL-6. TNF α levels were highest in +24h periods, and IL-6 in the +48h period. Serum CRP levels rose gradually peaking in the +72 hour period. While significant differences between Groups I and II were shown in the cytokines TNF α and IL-6, no difference was observed in CRP whose values did not differ with respect to BMI.

Figure 1 presents the dynamics of changes in serum visfatin levels in both groups with a maximum in periods +24 h. An analogous trend is documented in Figure 2 showing the dynamics of changes with leptin, also reaching a maximum in periods +24 h, although the statistical significance of the difference between the two groups is smaller (p < 0.01 vs p < 0.02). The different dynamics of changes in these adipokines and the so-called classic proinflammatory cytokines TNF α and IL-6 is evidence in Figures 3 and 4. With both cytokines, but with IL-6 in particular, the maximum of increase is shifted to the later time periods, primarily +48 h with IL-6. These increased levels persist longer, even in periods +72 h. a different dynamics of changes was noted with the typical marker of bacterial infection CRP. Figure 5 documents levels increasing with time, simultaneously in either group, regardless of the difference in BMI. The highest values were reached in the last measured period +72 h.

When comparing the dynamics of changes in visfatin and the other proinflammatory cytokines investigated, the highest regression coefficient of leptin was found in the +24h period (r=0.45; p < 0.02), whereas the correlations with the other inflammatory markers (TNF α, IL-6) were less significant (r=0.32 and r=0.20, respectively; p < 0.05). The relation to CRP was not significant as was that not to adiponectin in our study.

Discussion

The response of the body to surgical stress is characterized by a variety of inflammatory, hormonal, and metabolic changes, which in altogether produce the picture of acute phase reaction (5). The stress-producing stimulus of surgery inducing the release of a number of hormones (ACTH, cortisol, ADH, etc.) and proinflammatory cytokines (e.g., TNF α and IL-6) stimulates the system producing acute phase proteins in the liver, muscle catabolism, thermogenesis, and numerous other metabolic, psychological, and immunological responses (22).

The initiation, course and prognosis of the systemic inflammatory response to surgery thus depends on the interaction of proinflammatory and anti-inflammatory cytokines and other soluble and membrane mediators. Adipose tissue represents an important endocrine organ producing an array of hormones and cytokines including proinflammatory ones (e.g., TNF α and IL-6 among others). In obese patients, the rate of production of proinflammatory cytokines is generally higher, which may significantly interfere also with insulin metabolism (insulin resistance). Experimental and clinical studies have demonstrated that adipose tissue is infiltrated with immunocompetent cells, important producers of proinflammatory cytokines.

Visfatin (24) is a relatively new adipocytokine having, in addition to its effect on energy metabolism, a role in innate immunity and inflammation. Recent studies have documented in proinflammatory up to tissue-destroying effects (14), thus identifying it as an acute phase adipocytokine. Our study was designed to investigate also other adipocytokine (leptin, resistin, adiponectin) and classic proinflammatory cytokine (TNF α, IL-6, and CRP). The follow-up period (0h, +12h, +24h, +48h, and +72 hours) is consistent with the dynamics of cytokine expression in the early postoperative period. Investigations of clinical and laboratory inflammatory demonstrated only marginal differences in both of our groups – higher values of fating glucose, levels of insulin, triglycerides, and BP in Group I with BMI 31.2± 4.2 kg/m²; p < 0.05. There is little doubt this finding is related to the presence of higher amounts of visceral fat and insulin resistance. The higher proportion of adipose tissue also seems to be related with the not high, yet significantly higher levels of IL-6 and the decrease in adiponectin levels in the preoperative period in Group I.

The early postoperative period is associated with a rapid rise in visfatin levels reaching three-fold values within 24 hours (p < 0.01). This increase can be detected already at 12 hours (p < 0.02) whereas a gradual decrease does not occur until 48 hours (p < 0.02) to be followed by a return to a non-significant increase at 72 hours. During the course of our study, there were significant differences between the two groups, with Group II with BMI 27.3±3.3 kg/m² showing visfatin levels at all time periods, as documented in Figure 1. A analogous dynamics of changes was observed when monitoring leptin levels, also peaking as early as 24 hours postoperatively (sixfold and fourfold increases in Group I and Group II, respectively), Figure 2. In the case of visfatin, no relevant experimental and clinical data relate directly to the surgical procedure. With leptin, it has been shown (23) that, in the early postoperative period, surgical stress results in a transient elevation in leptin whose levels subsequently correlates with those of the proinflammatory cytokines IL-6 and TNF α, albeit with an earlier onset of changes. As our study documented analogous temporal dynamics of changes in visfatin and leptin, with a documented significant positive correlation (r=0.43; p < 0.02), it may be speculated there is analogous expression (both in time and quantity) of both adipose tissue adipocytokines in the early perioperative period. At the same time, we confirmed a perioperative increase in the proinflammatory cytokines TNF α and IL-6 with a maximum of changes in the later period. C-reactive protein showed a markedly later dynamics, with relatively high values persisting even after study completion. The levels of the “protective” adiponectin were highest prior to the onset of surgical stress to subsequently decline with inter-group differences of borderline significance.

The reported findings of dynamics of the two investigated cytokines (early increase)  are most important as, when compared with the dynamics of changes of the “classic” proinflammatory cytokines, the changes were observed later and differences in the inflammatory response were not related to the amount of adipose tissue. As a result, serum visfatin monitoring could serve as an early marker of inflammatory changes in the perioperative period also indicating an increased risk of expression of inflammatory cytokines in connection with the present visceral adipose tissue (unlike leptin, visfatin is expressed only by visceral adipose tissue). In the setting of inflammatory complications, CRP-based diagnosis could be delayed by as long as 48 hours compared with early diagnosis using selected adipocytokines, and visfatin in particular. However, its predictive value in terms of a longer-term inflammatory response is yet to be established.

The present study has its limitations, most importantly due to its small number of patients. Further, no more accurate anthropometric examination to predict the amount of visceral fat was performed (BMI is a most inaccurate measure in this respect). Other potential limitations may include pre- and post-operative management of  diabetes or hyperglycemia, as insulin and visfatin share one receptor, with the implication insulin may directly affect visfatin levels (however, maximum short-term insulin doses were not in excess of 20 U/day in our study).

Conclusion

In our study, significantly increased levels of the adiponectin visfatin, which correlated with leptin levels and were higher in the group of patients with higher BMI (visceral obesity) in the early postoperative period after abdominal surgery. The levels began to decrease gradually after 72 hours. The postoperative rise in classic proinflammatory cytokines (TNF α and IL-6) was delayed by 24–48 hours. The dynamics of increasing CRP levels was gradual with an increase persisting at the last examination at 72 hours, without a significant difference between the two groups.

Given its dynamics, visfatin could serve as an important predictor of early inflammatory changes in patients after abdominal surgery, in particular those with visceral obesity.

Abbreviations

BMI     -  body mass index

TNF α -  tumor necrotic factor alpha

IL-6    -  interleukin 6

CRP    -  C-reactive protein

NAD   -  nicotinamide adenin dinucleotide

IL-8    -  interleukin 8

IL-10  -  interleukin 10

DM     -  diabetes mellitus

ACTH -  adrenocorticotropic hormone

ADH   -  antidiuretic hormone

Supported by project (Health Minstry) for development of a research organization 00023001 (IKEM) – Institucional support

Address for correspondence:

MUDr Vladimír Teplan

Chirurgická klinika 3. LF UK a FN Královské Vinohrady

Šrobárova 50

100 00 Praha 10

Czech Republic

e-mail: vteplan@gmail.com