Serum Visfatin Increases With Progressive β-Cell Deterioration

One hundred and eighteen nondiabetic men, consecutively enrolled in a prospective study of cardiovascular risk factors in our health area, were included in the present study. None of these participants had evidence of metabolic disease other than nonmorbid obesity. Indeed, type 2 diabetes was ruled out by an oral glucose tolerance test (OGTT) according to criteria from the American Diabetes Association. Exclusion criteria for this group were 1) BMI ≥40 kg/m² and 2) concurrence of any systemic disease or medication use.

Sixty-four (35 men and 29 women) type 2 diabetic patients, defined according to the above-mentioned American Diabetes Association criteria and prospectively recruited from diabetes outpatient clinics at the Girona Hospital, were also studied. All type 2 diabetic patients had stable metabolic control in the previous 6 months. Pharmacological therapy for these subjects included insulin (34%), oral hypoglycemic agents (48%), statins (41%), fibrates (9%), blood pressure–lowering agents (48%), aspirin (27%), and allopurinol (5%). Exclusion criteria for this group were 1) clinically significant hepatic, renal, neurologic, endocrinologic, or other systemic disease, including malignancy; 2) current clinical evidence of hemochromatosis; and 3) acute major cardiovascular event in the previous 6 months. Type 1 diabetic patients with long-standing disease (n = 58; 31 men and 27 women; mean duration of diabetes 16 years [range 6–46]) and daily insulin doses >0.7 units/kg were also included in the study. Three of these patients had renal disease, and 12 patients exhibited retinopathy.

All subjects were of Caucasian origin, reported that their body weight had been stable for at least 3 months before the study, and were free from intercurrent illnesses or chronic infections at time of the study. Informed written consent was obtained after the purpose, nature, and potential risks of the study were explained to the subjects. The experimental protocol was approved by the ethics committee of the Hospital of Girona.

Subjects were studied in the postabsorptive state. BMI was calculated as weight in kilograms divided by the square of height in meters. Subjects’ waists were measured with a soft tape midway between the lowest rib and the iliac crest; hip circumference, when available, was measured at the widest part of the gluteal region; and waist-to-hip ratio was accordingly calculated. Blood pressure was measured in the supine position on the right arm after a 10-min rest. A standard sphygmomanometer of appropriate cuff size was used, and the first and fifth phases were recorded. Values used in the analysis are the average of three readings taken at 5-min intervals.

Insulin sensitivity (S_i), insulin secretion (acute insulin response to glucose [AIRg]), and glucose effectiveness (S_g) were measured by frequently sampled intravenous glucose tolerance tests (FSIGTs) in nondiabetic and newly diagnosed type 2 diabetic subjects. In brief, the experimental protocol for FSIGT studies started between 8:00 and 8:30 a.m. after an overnight fast. A butterfly needle was inserted into an antecubital vein, and patency was maintained with a slow saline drip. Basal blood samples were drawn at −30, −10, and −5 min, after which glucose (300 mg/kg body wt) was injected over 1 min starting at time 0, and insulin (0.03 units/kg Actrapid; Novo Nordisk, Bagsværd, Denmark) was administered at time 20 min. Additional samples were obtained from a contralateral antecubital vein up to 180 min, as previously described (7).

HDL cholesterol was quantified after precipitation with polyethylene glycol at room temperature. Total serum triglycerides were measured through the reaction of glycerol-phosphate-oxidase and peroxidase. Serum glucose concentrations were measured in duplicate by the glucose oxidase method using a Beckman Glucose Analyzer II (Beckman Instruments, Brea, CA). The coefficient of variation (CV) was 1.9%. Serum insulin concentrations were measured in duplicate by a monoclonal immunoradiometric assay (IRMA; Medgenix Diagnostics, Fleunes, Belgium). Intra- and interassay CVs were <7%. Serum visfatin concentrations were measured by an enzyme immunoassay (Phoenix Peptides, Karlsruhe, Germany). Sensitivity of the method is 0.1 ng/ml. The intra- and interassay CVs were <6%. HbA_1c (A1C) was measured by high-performance liquid chromatography (Bio-Rad, Muenchen, Germany) and a Jokoh HS-10 autoanalyzer.

Statistical analyses were performed using SPSS 12.0 software. Unless otherwise stated, descriptive results of continuous variables are expressed as means ± SD for Gaussian variables and as median (interquartile range) for non-Gaussian variables. Parameters that did not fulfill normal distribution were mathematically transformed to improve symmetry for subsequent analyses. The relation between variables was analyzed by simple correlation (Pearson’s test) and multiple regression in a stepwise manner. Unpaired Student’s t test was used to seek differences between men and women. One-way ANOVA followed by ANCOVA using general linear models (in order to correct for effect modifiers, such as sex, age, BMI, or A1C) were used to seek differences in plasma visfatin concentrations among groups. Levels of statistical significance were set at P < 0.05. For a given value of P = 0.05, the study had an 80% power to detect significant correlations (Pearson’s coefficient of at least 0.3) between serum visfatin and metabolic parameters in bilateral tests. The study was also powered to detect a significant difference in circulating visfatin of at least 0.5 SD between nondiabetic and type 2 diabetic subjects.

Clinical and biochemical variables of the study subjects are summarized in Tables 1 and 2. Circulating visfatin was significantly associated with AIRg (r = −0.27, P = 0.002) in nondiabetic men (Fig. 1). Similarly, circulating visfatin was negatively correlated with 30-min OGTT insulin in these subjects (r = −0.24, P = 0.005). No significant correlations were found between visfatin and age, BMI, waist circumference, S_g, A1C, and 2-h OGTT plasma glucose in nondiabetic subjects. Serum visfatin was also significantly correlated with fasting insulin (r = −0.20, P = 0.042) and S_I (r = 0.19, P = 0.043) in nondiabetic men. However, on stepwise multiple regression analyses, AIRg was the only predictor of circulating visfatin, explaining 8% of its variance in nondiabetic men (Table 3).

As previously shown (4), serum visfatin was found to be increased in type 2 diabetes (mean 18 [95% CI 16–21] vs. 15 ng/ml [13–17] for type 2 diabetic and nondiabetic subjects, respectively; P < 0.0001 nonadjusted, P = 0.017 adjusted for sex, age, and BMI). In these subjects, A1C was highly correlated with circulating visfatin (r = 0.49, P < 0.0001; Fig. 2), and adjusting for A1C significantly attenuated the difference in serum visfatin between type 2 diabetic and nondiabetic subjects (17 [15–20] vs. 16 ng/ml [14–19], respectively; P = 0.46 adjusted for sex, age, BMI, and A1C).

To get further insight into the association of plasma visfatin with type 2 diabetes, subjects were divided into known (n = 47) and newly diagnosed (by OGTT) (n = 17) type 2 diabetic patients (Fig. 3). Known type 2 diabetic, in contrast to newly diagnosed type 2 diabetic, subjects have increased A1C values (6.9 ± 1.3 vs. 5.0 ± 0.9 vs. 4.9 ± 0.4% for known type 2 diabetes, new type 2 diabetes, and no diabetes, respectively; P < 0.0001).

By studying only known type 2 diabetic subjects, we documented persistently increased circulating visfatin in this metabolic disease, after accounting for the effect of the above-mentioned covariates (mean 19 [95% CI 16–22] vs. 15 ng/ml [13–17] for known type 2 diabetes and nondiabetic subjects, respectively; P < 0.05; Fig. 3). Newly diagnosed type 2 diabetes, on the other hand, exhibited circulating visfatin values that were similar to those of nondiabetic subjects (15 [10–19] vs. 15 ng/ml [13–17], respectively; P = NS, adjusted for sex, age, BMI, and A1C; Fig. 3). In accord with their A1C values, the latter patients showed only a partial defect in β-cell function, as evidenced by detectable AIRg values in most of these subjects (144 [0–343] vs. 444 [386–502] for new type 2 diabetic and nondiabetic subjects, respectively; P = 0.004 adjusted for sex, age, BMI, and A1C). On multiple regression analysis, A1C, but not sex, age, or BMI, was a predictor of circulating visfatin in type 2 diabetic patients, explaining 20% of its variance (β = 0.457, P < 0.0001).

To further test whether serum visfatin is dependent on preserved β-cell function, a group of type 1 diabetic patients with long-standing disease (n = 58; mean duration of diabetes 16 years [range 6–46]) were also studied (Table 2). Circulating visfatin was increased in type 1 diabetic vs. type 2 diabetic and nondiabetic subjects (37 [34–40] vs. 17 [15–20] and 16 ng/ml [14–19], respectively; P < 0.0001 adjusted for sex, age, BMI, and A1C compared with either type 2 diabetic or nondiabetic subjects; Fig. 3). Circulating visfatin, however, was unrelated to hyperglycemia (a potential confounding factor in the association between visfatin and β-cell function), as evidenced by a lack of correlation between serum visfatin concentration and A1C in type 1 diabetic subjects (r = 0.17, P = 0.20; Fig. 2B). On multiple regression analysis, age, but not sex, BMI, or A1C, was a predictor of circulating visfatin in type 1 diabetic patients, explaining 10% of its variance (β = 0.344, P = 0.010).

To our knowledge, this is the first report of an association between serum visfatin and β-cell dysfunction. Visfatin was recently shown to play a role in the amelioration of hyperglycemia in two different animal models of diabetes (2). Both overexpression of visfatin gene and treatment with recombinant protein exerted blood glucose–lowering effects in diabetic mice. Of note, recombinant visfatin was equipotent to exogenous insulin in this effect (2). To further support these findings, deletion of one of the two copies of the visfatin gene caused defects in glucose tolerance in mice (2). These observations, together with reports of increased serum visfatin in type 2 diabetes in both mice and humans (2,4), point to a potential relevant role of this novel adipocytokine in either the pathogenesis or the clinical phenotype of type 2 diabetes.

Recent efforts (3,4,8,9) to elucidate the role of visfatin in this disease have failed to describe an association of the adipocytokine with insulin sensitivity. Not surprisingly, visfatin was inconsistently related to anthropometrical and biochemical surrogates of insulin resistance, such as BMI, waist circumference, abdominal fat, fasting insulin, HOMA-IR, or serum adiponectin, in these and other studies (5,10). Similarly, genetic variations in the visfatin gene were only modestly associated with diabetes-related parameters, such as 2-h plasma glucose and fasting insulin concentration (11). Yet, the type 2 diabetes phenotype requires also a defect in β-cell function (12), which appears to has been overlooked in prior investigations.

Although insulin is unlikely to directly regulate visfatin serum concentrations (no effects on visfatin synthesis were documented after treating 3T3-L1 cells with insulin [13], no changes in plasma visfatin were observed upon fasting or feeding in mice [2], no differences in serum visfatin were seen in type 2 diabetic subjects treated with sulfonylureas or exogenous insulin [4] and our own data [not shown], and no independent associations were observed between fasting insulin and serum visfatin in our study), our results are consistent with a physiopathological mechanism linked to β-cell dysfunction that mediates the association between increased circulating visfatin and both type 2 and type 1 diabetes. Besides its negative association with AIRg from FSIGT, serum visfatin was also negatively correlated with 30-min OGTT insulin (a surrogate index of insulin secretion) in our study. Furthermore, serum visfatin was recently shown to decrease following weight loss in morbidly obese subjects (10), an intervention that is believed to improve β-cell function (14,15). It should be noted that this mechanism appears not to be operative in the early phases of β-cell deterioration, as both glucose-intolerant and newly diagnosed type 2 diabetic subjects did not have increased serum visfatin in our study. A more severe defect, as seen in long-standing diabetes, is therefore needed to dysregulate circulating visfatin.

Circulating visfatin was also highly correlated with A1C in type 2 diabetic patients. Although factors related to hyperglycemia or oxidative stress can mediate in part the association between visfatin and diabetes (as indicated by attenuation of the difference in circulating visfatin between nondiabetic and type 2 diabetic subjects after adjusting for A1C), the fact that this difference persisted in known type 2 and in type 1 diabetic subjects, together with a lack of association between plasma visfatin and A1C in type 1 diabetic subjects, indicate that other factors besides hyperglycemia are involved in this relationship. On the other hand, increased A1C may reflect a more severe β-cell dysfunction (16), and, thus, adjusting for this factor will not surprisingly decrease the association of circulating visfatin with diabetes.

Among the counterregulatory hormones that are dysregulated in diabetic patients, both growth hormone and catecholamines were shown to be negative regulators of visfatin expression (13). Glucocorticoids, on the other hand, are known to induce visfatin secretion in adipocytes (13), and thus could contribute to the increased concentrations of this adipocytokine that accompany diabetes. It is also likely that other hormones of the entero-insular axis upregulate serum visfatin in the course of β-cell deterioration.

More recently, Haider et al. (17) have reported on the effects on glucose and insulin on circulating visfatin in vivo and on visfatin secretion in vitro. Their results support our findings in type 2 diabetes by showing an upregulation of visfatin by hyperglycemia both in vivo and in vitro. Although insulin administration prevented the rise in visfatin concentrations under these conditions, insulin alone had no effect on either plasma visfatin or visfatin expression. Furthermore, other factors besides insulin must be involved in the regulation of plasma visfatin, as somatostatin, a general inhibitor of hormone secretion, was equipotent to insulin in preventing the rise in circulating visfatin following hyperglycemia in vivo (17).

As to the role of visfatin in human diabetes, insulin-mimetic actions are to be expected, as supported by the experimental animal data (2), and thus the increased serum visfatin concentration can respond to a compensatory mechanism aimed at ameliorating the functional consequences of insulin deficiency. The increased visfatin concentration may also promote insulin sensitivity by its stimulatory effects on peroxisome proliferator–activated receptor-γ and adiponectin gene expression (2). Yet, both unresponsiveness to visfatin actions (as seen for insulin and leptin) and a contribution of visfatin to β-cell deterioration cannot be ruled out in diabetic patients. In summary, circulating visfatin is increased with progressive β-cell deterioration. The study of the regulation and role of visfatin in diabetes merits further consideration.

This study was supported, in part, by grants PI041407 (to A.L.-B.), PI041383, G03/212, and G03/028 (to J.-M.F.-R.) from the Fondo de Investigación Sanitaria, Carlos III Health Institute, Spain, and partly funded by grant BFU2004-03654/BFI (to J.-M.F.-R.) from the Ministry of Education and Science, Spain. A.L.-B. is also a Research Investigator of the Fund for Scientific Research “Ramon y Cajal” (City for Ministry of Education and Science of Spain, Madrid).