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Hindawi Publishing CorporationJournal of ObesityVolume 2015, Article ID 291209, 8 pages Clinical Study
Tissue Factor Expression in Obese Type 2 Diabetic Subjects and
Its Regulation by Antidiabetic Agents
Jing Wang,1,2 Theodore P. Ciaraldi,3,4 and Fahumiya Samad1,2
1 Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, USA2San Diego Biomedical Research Institute, San Diego, CA 92121, USA3VA San Diego Healthcare System, San Diego, CA, USA4Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA Correspondence should be addressed to Fahumiya Samad; [email protected] Received 15 November 2014; Revised 17 February 2015; Accepted 19 February 2015 Academic Editor: Aron Weller Copyright 2015 Jing Wang et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Objective. Increased coagulation activation may contribute to the high incidence of cardiovascular complications observed in obeseand type 2 diabetes (T2D) subjects. Although tissue factor (TF), the primary initiator of coagulation is increased in obesity, itsexpression in adipose tissues and its association with metabolic parameters are unclear. We sought to compare TF expression inplasma and adipose tissues of obese subjects with and without T2D, its correlation with metabolic parameters, and regulation inresponse to antidiabetic drugs. Methods Subjects were recruited from diabetes clinics and adipose tissue was obtained by needlebiopsy of lower subcutaneous abdominal depot. For the intervention study, subjects were randomized into treatment groups withrosiglitazone or metformin for 4 months. Results. Plasma TF antigen, activity, and adipose TF mRNA were greater in obese T2Dsubjects compared with obese nondiabetics. Plasma TF activity correlated with fasting insulin, glucose, and free fatty acids, (FFAs),and adipose TF mRNA correlated with plasma FFA. Plasma TF activity was reduced by metformin and increased with rosiglitazonetreatment. Conclusions. Specific diabetes-related metabolic parameters, but not obesity per se, are correlated with TF expression.
Regulation of TF activity by different classes of antidiabetic drugs may relate to protective or adverse cardiovascular outcomes.
TF-FVIIa complex triggers extrinsic coagulation leading toFXa-mediated generation of the downstream coagulation Obesity is a major risk factor for the development of type protease thrombin, fibrin deposition, and platelet activation.
2 diabetes (T2D), and clinical studies have established an Higher plasma concentrations of FVII [3], increased levels increased incidence of thrombosis and cardiovascular disease of thrombin and thrombin-antithrombin (TAT) complexes as a primary cause of mortality in diabetic patients [1, 2]. T2D [4], and increased circulating monocyte TF procoagulant is associated with accelerated and premature atherosclerosis activity [5] are observed in obese subjects. Weight loss as well as other cardiovascular and thrombotic complica- in morbidly obese patients significantly reduces thrombin tions including myocardial infarction, ischemic stroke, and generation potential [6] and decreases levels of circulating peripheral vascular disease [1, 2]. While risk factors such TF, FVII, and prothrombin fragment F1.2, a marker of in vivo as hypertension and cholesterol are elevated in T2D, these thrombin formation [7]. Compared with nondiabetic control only account partially for the cardiovascular burden, and subjects, T2D patients display signs of hypercoagulability and emerging evidence suggests that dysregulation of pathways increased plasma TF procoagulant activity [8, 9], increased of coagulation is an additional significant mechanism that abundance of TF-positive microparticles [8, 10], and higher contributes to increased cardiovascular risk.
TF activity of circulating monocytes [11, 12]. Elevated blood- TF is the primary initiator of the coagulation pathway borne or circulating TF correlates with microvascular com- and is the cell surface receptor for coagulation factors VII plications and is considered a biomarker for the severity of and VIIa. Cell surface or microparticle- (MP-) associated microvascular disease in T2D patients [13, 14].
Journal of Obesity Whereas plasma TF activity and antigen levels have Table 1: (a) Metabolic characteristics of subjects and (b) baseline been shown to be increased in obese subjects as well as parameters of subjects prior to treatment with metformin or rosigli- in patients with T2D, whether its expression is higher in obese diabetics compared with obese nondiabetics is unclear.
This is especially important since all obese subjects are notnecessarily diabetic. In genetically or high fat diet induced obese mice that are also diabetic, TF activity is increased in the blood and its activity and gene expression increased in adipose tissues [15–17]. However, to our knowledge whether adipose TF expression is similarly induced in obese and diabetic humans has not been studied. Here, we examined Fasting glucose (mmol/L) TF expression in plasma and adipose tissues of obese subjects Fasting insulin (pmol/L) with and without diabetes, how its expression correlates with a number of metabolic parameters, and how its activity isregulated in response to the antidiabetic drugs, metformin, and rosiglitazone.
2. Patients and Methods
Human subject protocols were approved by the Committee on Human Investigation at the University of California, San Diego (UCSD), and all subjects provided written informed Fasting glucose (mmol/L) consent. Subjects were recruited from diabetes clinics and Fasting insulin (pmol/L) classified as diabetic or nondiabetic by their response to a 75 g oral glucose tolerance test according to the American Diabetes Association criteria and classified as obese if their BMI (kg/m2) > 30 as previously described [18]. Adipose tissue was obtained by needle biopsy of the lower subcutaneous For Tables 1(a) and 1(b), data are means ± SD. TG, triglyceride.
abdominal depot [18]. Other clinical data are summarized in Table 1(a): ∗𝑃 < 0.05; ND versus T2D.
Table 1(a). The time from initial diabetes diagnosis to samplecollection ranged from 0.2 to 13 years. None of the subjectshad a history of thromboembolism. All routine laboratory parameters for subjects in Table 1 were determined using test values were within 2.5X the upper and lower limits of standard techniques as previously described [18, 19].
specified normal ranges. A majority of the T2D subjects were Plasma TF antigen was determined using the IMUBIND not taking any antidiabetic medications at the time of biopsy Tissue Factor ELISA kit, and TF procoagulant activity in and were controlled by diet alone as part of a 6-week washout plasma was determined using the chromogenic Actichrome of antidiabetic medications. Of the other T2D subjects, 1 was TF activity assay according to manufacturer's instructions on glucovance and 1 was taking rosiglitazone and the other (American Diagnostica, Stamford, CT). Adipose TF gene glyburide and metformin at the time of biopsy.
expression was determined by real-time quantitative RT-PCR In the intervention study (Table 1(b)), subjects were as previously described [17]. cDNA synthesized from total excluded if they were previously treated with TZD, treated RNA was analyzed with gene specific primers (Invitrogen) with more than one diabetic agent or insulin, had hyper- and SYBR Green PCR Master mix (PerkinElmer) in an tension, were pregnant, or had active cardiac disease or iCycler (Bio-Rad). Relative gene expression levels were cal- other major illnesses. If applicable, subjects maintained culated after normalizing to 𝛽-actin using the ΔΔCT method their blood pressure and lipid-controlling medications. There (Bio-Rad). Statistical analysis was evaluated using the Graph- were no differences at baseline between treatment groups Pad Prism program (Intuitive Software, San Diego, CA).
with regard to duration of diabetes, blood pressure, rou- The statistical significance of differences within and between tine laboratory values, or use of blood pressure or lipid- groups was analyzed by the Student's paired and unpaired controlling medications. Briefly, after a washout period of 𝑡-tests. Correlations were calculated using the Pearson cor- 6 weeks for those participants that were on an antidiabetic relation. A 𝑃 value < 0.05 was considered as the level of treatment, subjects were randomized into a treatment group with high-dose rosiglitazone (4 mg twice daily) or high-dosemetformin (1,000 mg twice daily) for a period of 4 months 3. Results and Discussion
[19]. To minimize potential side effects, medications wereinitiated below target doses and titrated up over the initial Although obesity poses a risk for the development of insulin 2 weeks. Both subjects and investigators were blinded to resistance and T2D, not all obese subjects are diabetic. The treatment. Blood was collected after 10–12-hour overnight influence of T2D on tissue factor expression in obese subjects fast at baseline and 4-month posttreatment. All metabolic was evaluated in cohorts matched for weight and BMI as Journal of Obesity Figure 1: Plasma TF activity (a), antigen (b), and adipose TF mRNA levels (c) in obese T2D subjects compared with obese nondiabetics. Forpanels (a) and (b), N = 10–13 ± SD. For panel (c), N = 6–10 ± SD. ∗∗𝑃 < 0.01 nondiabetics versus T2D. Clinical characteristics of subjects areindicated in Table 1(a).
indicated in Table 1(a). The body weights (kg) of obese Table 2: Correlation between TF and metabolic parameters.
nondiabetics and diabetics were 105 ± 17.3 and 101 ± 15.9,while their BMIs (kg/m2) were 35.6 ± 5.6 and 35.6 ± 7.1, TF activity TF antigen Adipose TF mRNA respectively. Fasting levels of plasma glucose, insulin, free fatty acids (FFAs), and HbA1c were significantly higher in the obese diabetic cohorts, while no differences were observed for plasma triglycerides between the two groups. Plasma TF activity, plasma TF antigen, and adipose TF mRNA expression were significantly increased in obese diabeticscompared with obese nondiabetics (Figures 1(a)–1(c)).
In the commercial Actichrome TF activity assay (Ameri- can Diagnostica) plasma samples are directly incubated with FVIIa, FX, and a chromogenic substrate for FXa. A limitation of this assay is that the values obtained tend to be somewhat overestimated. However, regardless of the absolute levels of ∗𝑃 < 0.05, ∗∗𝑃 < 0.01, ∗∗∗𝑃 < 0.001 for TF (activity, antigen or adipose TF activity, we believe that, on a relative basis, the observation mRNA) versus indicated metabolic parameters.
that plasma TF activity is higher in obese T2D relative toobese nondiabetics is relevant. In the plasma, TF exists in theform of microparticles shed from apoptotic or activated cells, Since metabolic changes observed in T2D may contribute as well as an alternately spliced soluble form of TF. It should to elevated TF expression, we determined the correlation be noted that the TF activity and antigen assays used did not of TF expression with a number of metabolic parameters.
discriminate between these various forms of TF but rather As indicated in Table 2, no significant correlations were measured total plasma TF.
observed for plasma TF activity and antigen with either BMI Journal of Obesity Fasting glucose (mmol/L) Fasting insulin (pmol/L) Fasting insulin (pmol/L) Figure 2: Correlations between (a) plasma TF activity levels and plasma fasting glucose, (b) plasma TF activity and fasting insulin, (c)plasma TF antigen and fasting insulin, (d) plasma TF activity and plasma free fatty acid (FFA), (e) plasma TF antigen and plasma FFA, and(f) adipose TF mRNA and plasma FFA. Correlations were calculated using the Pearson correlation. A 𝑃 value < 0.05 was considered as thelevel of significance.
or weight suggesting that obesity per se may not contribute to and FFA (Table 2, Figure 2). Interestingly adipose TF mRNA elevated TF activity. Alternately, TF activity was significantly was correlated only with plasma FFA (Table 2, Figure 2(f)).
correlated with fasting glucose, insulin, and free fatty acid Hyperinsulinemia is associated with insulin resistance (FFA), while TF antigen was correlated with fasting insulin and T2D, and insulin administration has been demonstrated Journal of Obesity to increase circulating TF in healthy and T2D subjects [9, were observed in metformin-treated T2D patients and in 20] and in obese insulin-resistant and diabetic mice, insulin prediabetics on statin and metformin therapy [27]. To directly increases both circulating and adipose TF expression [15, 16].
determine in vivo efficacy of metformin on suppressing Microparticles shed from activated monocytes are important coagulation, we determined the effect of metformin on TF sources of blood-borne TF. Compared with normal mono- activity in obese T2D subjects. Baseline characteristics of cytes, insulin-resistant T2D monocytes express more TF and the treatment group are summarized in Table 1(b). Four- shed MP with a higher TF levels in response to insulin [11].
month high-dose metformin treatment led to a moderate TF expression is negatively regulated by phosphoinositide 3- insignificant reduction of plasma TF activity (Figures 3(a) kinase signaling [21], suggesting that downregulation of this and 3(e)). Out of eight patients that were followed, six pathway as a consequence of insulin resistance may favor TF showed a reduction in TF activity, while in two of the upregulation in T2D [11]. Our study indicating that plasma subjects TF activity was modestly increased (Figure 3(b)).
TF activity and antigen are correlated with insulin levels is These small decreases in TF activity observed in 75% of the consistent with these previous data and suggests that the patients however may be clinically relevant as such changes compensatory hyperinsulinemia that accompanies insulin in hemostatic factors including factor VIIa have been shown resistance and T2D may contribute to the observed increase to determine cardiovascular risk [28].
in plasma TF expression in these patients. A significant cor- Thiazolidinediones (TZDs) that act through the nuclear relation was also observed between TF activity and glucose, and this is in line with data indicating that hyperglycemia (PPAR𝛾) are potent insulin sensitizers and highly effective in healthy volunteers and T2D increases TF procoagulant oral medications for T2D [29]. However, clinical trials have activity [9] and improved glycemic control reduces circulat- indicated that the TZD and rosiglitazone (Avandia) lead to a ing TF [22]. Importantly, combined hyperinsulinemia and high risk of myocardial infarction and cardiovascular disease hyperglycemia produced an additive increase in TF activity leading to their withdrawal [29]. While the reason for its [20], suggesting that the presence of these two conditions adverse cardiovascular side effects is not fully understood, that often occur concurrently in T2D may contribute to the in vitro studies have demonstrated that rosiglitazone upreg- observed increase in TF and procoagulant activity in thesepatients.
ulated the expression of procoagulant TF bearing MP byhuman monocytes/macrophages [30]. To determine if In this study, the metabolic parameter that showed the rosiglitazone regulates TF activity in vivo, we measured strongest correlation with plasma TF activity and antigen and plasma TF activity in a cohort of obese T2D subjects at the only correlate associated with adipose TF expression was baseline and after 4 months of rosiglitazone treatment.
FFA (Table 2, Figure 2(f)). Obesity is associated with a dyslipi-demia that in part is manifested as increased circulating FFA.
A significant increase in TF activity was observed after Increased adipose lipolysis due to insulin resistance is a pri- rosiglitazone treatment compared with baseline levels mary mechanism leading to elevated FFA in the plasma and (Figures 3(c) and 3(e)). Of the 10 patients that were studied, in the local microenvironment of obese T2D patients. FFA 7 of them showed an increase and 3 showed a decrease activates toll-like receptor- (TLR-) mediated signaling, and in in plasma TF activity (Figure 3(d)). To our knowledge, monocytes induction of TF expression has been shown to be this is the first in vivo demonstration where rosiglitazone dependent on the coordinated activation of TLR-dependent induces TF activity in human subjects which may potentially nuclear factor-𝜅B (NF-𝜅B) and Jun N-terminal kinase (JNK) contribute to the adverse cardiovascular outcomes with this signaling pathways [21]. It is tempting to speculate that FFAs drug. The mechanism for rosiglitazone-mediated increase in could similarly increase TF expression in adipocytes and TF activity is unclear and is not related to metabolic measures adipose tissue macrophages via TLR-mediated activation of of insulin sensitivity as these were markedly improved in NF-𝜅B and JNK signaling cascades. A role for FFA in TF rosiglitazone-treated patients [19]. Despite their side effects, induction was confirmed by our finding that TF expression TZDs are still considered to have therapeutic value due to in adipocytes is increased by palmitate (Samad, unpublished their superior antidiabetic effects. Since increased TF activity observation), the most abundant plasma FFA in obesity.
was not observed in all rosiglitazone-treated subjects, it is Metformin, a member of the biguanide family, is one of tempting to speculate that measures of TF activity could be the most commonly used antidiabetic drugs and is highly a useful indicator to identify patients that may be at risk for effective in lowering blood glucose and improving peripheral developing cardiovascular disease in response to TZDs.
insulin sensitivity in patients with T2D [23]. In addition, In conclusion, our study shows that plasma and adipose increasing studies indicate that metformin reduces the risk TF expression is increased in obese T2D compared to obese of diabetes-associated atherothrombotic disease independent nondiabetic subjects and significantly correlated with FFA.
of its antihyperglycemic effect [24]. A limited number of Regulation of TF activity by different classes of antidiabetic in vitro and clinical studies have suggested that the cardio- drugs may relate to protective or adverse cardiovascular protective effects of metformin may be related to beneficial effects on pathways of fibrinolysis and hemostasis. In vitrostudies show that metformin suppresses TF expression in Conflict of Interests
human monocytes and reduces thrombin activity and fibrinpolymerization [25, 26]. Modest reductions of VIIa activity The authors state that they have no conflict of interests.
Journal of Obesity Figure 3: Plasma TF activity at baseline and following treatment with metformin ((a), (b), and (e)) and rosiglitazone ((c), (d), (e)). The 𝑥-axisnumbers (1–8) in Figure 3(b) and (1–10) in Figure 3(d) represent each patient and are not identical as these are subjects from two treatmentsstudies. For metformin-treated subjects in panels (a), (b), and (e): N = 8 ± SD. For rosiglitazone-treated subjects in panels (c), (d), and (e):N = 10 ± SD. ∗𝑃 < 0.05, baseline versus rosiglitazone. Baseline clinical characteristics of subjects are indicated in Table 1(b).
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