Shedding of TNF-alpha receptors, blood pressure, and insulin sensitivity in type 2 diabetes mellitus

José-Manuel Fernandez-Real1,2, Begoña Lainez1,3, Joan Vendrell2, Mercedes Rigla4, Antoni Castro5, Georgina Peñarroja5, Montserrat Broch2, Antonio Pérez4, Cristobal Richart2, Pablo Engel3, and Wifredo Ricart1,2

1 Unitat de Diabetologia, Endocrinologia i Nutricio, 5 Servei de Medicina Interna, University Hospital of Girona, 17001 Girona; 2 Unitat de Endocrinologia i Nutricio, University Hospital of Tarragona; Institut d'Estudis Avançats, Tarragona; 3 Immunology Unit, Department of Cellular Biology and Pathology, Institut de Investigació Biomèdica August Pi Sunyer, Medical School, University of Barcelona, Barcelona; and 4 Servei d'Endocrinologia, Hospital de Sant Pau, Barcelona, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor-alpha (TNF-alpha ) is increasingly recognized as a key component in the development of insulin resistance and increased blood pressure. In a sample of 368 individuals, the ratio of soluble TNF-alpha receptors (sTNFR2/sTNFR1) correlated positively with systolic and diastolic blood pressure (P < 0.01). This ratio was significantly greater in type 2 diabetic subjects (DM-2) than in type 1 diabetic patients and was greater than in control nondiabetic subjects (P < 0.00001). The TNF-alpha receptor 1 (TNFR1) density in peripheral blood monocytes was similar in DM-2 patients and in nondiabetic subjects. After phorbol 12-myristate 13-acetate, TNFR1 shedding was significantly decreased in DM-2 compared with control subjects, and it was directly associated with insulin sensitivity (r = 0.54, P = 0.03). Serum sTNFR1 concentration was also linked to the vasodilatory response to glyceryltrinitrate (P = 0.01). Conversely, TNF-alpha receptor 2 shedding was negatively associated with insulin sensitivity (r = -0.54, P = 0.03), whereas shedding of L-selectin showed no significant association. After exercise-induced lowering of blood pressure, a parallel decrease in sTNFR2/sTNFR1 was observed in DM-2 patients. Our findings suggest that insulin resistance and blood pressure are linked to altered shedding of TNF-alpha receptors in DM-2. The latter seems reversible and is not genetically determined.

vascular dysfunction; exercise; flow cytometry; nitric oxide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HYPERTENSION is a major cause of morbidity and mortality in diabetic patients (5). The mechanism by which hypertension occurs is most likely multifactorial, based on the chronic metabolic disturbance. Immunopathogenic mechanisms are increasingly recognized to be involved in the pathogenesis of hypertensive disease in which alterations in both humoral and cellular immunity have been described (15, 18). Tumor necrosis factor-alpha (TNF-alpha ) is a proinflammatory cytokine that, in addition to its role in the immune response and cancer, is implicated in the phenotypic expression of insulin resistance (17). The complete absence of TNF-alpha results in a significant improvement in insulin sensitivity in mice with dietary, hypothalamic, or genetic obesity (reviewed in Ref. 17). In humans, recent studies suggest that the TNF-alpha gene locus is involved in insulin resistance-associated hypertension (27). A positive correlation between serum TNF-alpha concentration and both systolic blood pressure (SBP) and insulin resistance has been found in subjects with a wide range of adiposity (38). Upregulation of TNF-alpha secretion has been observed in peripheral blood monocytes from hypertensive patients (12). The available information on direct TNF-alpha effects also suggests that it is involved in the pathophysiology of hypertension. TNF-alpha stimulates the production of endothelin-1 (19) and angiotensinogen (7). In the spontaneously hypertensive rat model, TNF-alpha synthesis and secretion are increased in response to lipopolysaccharide (LPS) stimulation compared with those in nonhypertensive control rats. LPS increased fat angiotensinogen mRNA in the former but not in the latter (24). TNF-alpha is also thought to cause endothelial dysfunction linked to insulin resistance (37).

TNF-alpha binds to two TNF-alpha receptors, TNFR1 and TNFR2. Each receptor is expressed by most cells and can be regulated independently. After binding to its receptors, a proteolytic cleavage of the extracellular parts elicits the soluble forms, named (2, 22) sTNFR1 (55 kDa) and sTNFR2 (75 kDa). The soluble sTNFRs remain elevated for longer periods of time after the administration of TNF-alpha and are thought to measure previous TNF-alpha effects (2). sTNFRs were constantly found in the circulation of patients with sepsis, as a surrogate of the inflammatory state even when TNF activity was undetectable (34). Measurements of the sTNFRs' concentrations in healthy individuals at different time lapses showed that their concentrations in the same subject were quite stable, at least during a period of 1 yr (3).

Isolated limb perfusion with TNF-alpha to cancer patients led simultaneously to increased plasma sTNFR1 and sTNFR2 concentrations. Different mechanisms were responsible for shedding of TNFR1 and TNFR2 (4). The TNFR1 stimulation with excess TNF-alpha seemed to transactivate preferential TNFR2 shedding (17).

We have previously shown that plasma sTNFR2 concentration was associated with insulin resistance in nondiabetic subjects (16). We tested the hypothesis that the sTNFR2-to-sTNFR1 ratio (R2/R1), as a correlate of TNF-alpha action and insulin sensitivity, was associated with blood pressure in a sample of 368 individuals. We found that R2/R1 correlated positively with SBP and diastolic blood pressure (DBP; r = 0.144 and 0.14, P = 0.005 and 0.007). The R2/R1 was significantly greater in type 2 diabetic subjects (DM-2, n = 66) than in type 1 diabetic patients (DM-1, n = 46) and was greater than in control nondiabetic subjects (n = 258; 2.65 ± 1.05 vs. 2.24 ± 0.7 vs. 1.76 ± 0.38, ANOVA, P < 0.00001). In fact, the R2/R1 seems to be a good correlate of TNF-alpha action in subjects with insulin resistance (11). Given these findings, we hypothesized altered TNFR1 and TNFR2 shedding in insulin resistance-associated hypertension.

The objectives of this study were to evaluate whether TNFR1 and TNFR2 shedding was associated with insulin action and with vascular dysfunction, as a surrogate complication of insulin resistance, in DM-2 patients and to test whether shedding of TNF-alpha receptors changes after lowering of blood pressure.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Patients: Control Subjects

Two hundred fifty-eight subjects (109 women) were evaluated between July 1 and September 15, 1997 as a part of an ongoing epidemiological study dealing with the influence of inflammatory factors on body composition. None of the subjects was taking any medication or had any evidence of metabolic disease other than obesity. All subjects were of Caucasian origin and reported that their body weight had been stable for at least 3 mo before the study. Inclusion criteria were as follows: 1) body mass index (BMI, weight in kilograms divided by the square of height in meters) <40 kg/m2, 2) absence of any systemic disease, 3) absence of any infections in the previous month, and 4) SBP <165 mmHg. Resting blood pressure was measured with a mercury sphygmomanometer after subjects had been in a sitting position for a minimum of 15 min and was read three times in the right arm by the same investigator. The mean of three measurements was used for this study. Liver and renal diseases were specifically excluded by biochemical workup.

Inclusion and Exclusion Criteria for Diabetic Patients

Forty-six (21 women) consecutive DM-1 patients and 66 (20 women) DM-2 patients were prospectively recruited from diabetes outpatient clinics on the basis of the following criteria: 1) aged 40-70 yr, 2) current BMI <40 kg/m2, 3) stable metabolic control in the previous 6 mo, and 4) no history of ketoacidosis. Exclusion criteria included the following: 1) clinically significant hepatic, neurological, endocrinological, or other major systemic disease, including malignancy; 2) history of drug or alcohol abuse, defined as >80 g/day in men and >40 g/day in women, or serum transaminase activity over two times the upper limit of normal; 3) SBP >165 mmHg, previous antihypertensive therapy for uncontrolled hypertension and elevated serum creatinine concentration; 4) acute major cardiovascular event in the previous 6 mo; 5) acute illnesses and current evidence of acute or chronic inflammatory or infective diseases; and 6) mental illness rendering the subjects unable to understand the nature, scope, and possible consequences of the study. DM-1 patients were defined according to World Health Organization criteria (36) and, except for history of ketoacidosis, fulfilled the preceding criteria. All patients underwent a full medical history, including age, duration of diabetes, BMI, eating habits, smoking habits, blood pressure, total cholesterol, and a full examination to screen for diabetic complications. The experimental protocol was approved by the Ethics Committee of the Girona University Hospital. Informed written consent was obtained after the purpose, nature, and potential risks were explained to the subjects.

Definition of Chronic Diabetic Complications

The clinical diagnosis of diabetic retinopathy was based on the examination of the ocular fundus after dilatation of the pupils by experienced ophthalmologists. Simplex retinopathy was defined as one or more microaneurysms or hemorrhages. Diabetic macroangiopathy complications were diagnosed according to clinical findings, Doppler sonography, and angiopathy. Persistent microalbuminuria was defined as an albumin excretion rate of 30-300 mg/day.

Measurements

Each subject was studied in the research laboratory in the postabsorptive state. The room was quiet, lights were dimmed, and temperature was controlled at 23°C. Alcohol, caffeine, and all medications, including sulfonylurea, metformin, and insulin, were withheld within 12 h of the different tests.

Study of insulin sensitivity. After the intravenous injection of regular insulin, glucose levels were determined every minute during 15 min. Insulin sensitivity was indicated by the first-order rate constant for the disappearance rate of glucose (KITT) estimated from the slope of the regression line of the logarithm of blood glucose against time during the first 3-15 min.

Brachial artery vascular reactivity. High-resolution external ultrasound (128XP/10 mainframe with a 7.5-MHz linear array transducer; Toshiba SSH-140A) was used to measure changes in brachial artery diameter in response to 400 µg of sublingual nitroglycerin (NTG), an endothelium-independent, direct smooth muscle dilator, as described by Celermajer et al. (9). The lumen diameter of the artery was defined as the distance between the leading edge of the echo of the near wall-lumen interface to the leading edge of the far wall-lumen interface echo. All scans were ECG-controlled for the R wave. All images were recorded on a super-VHS videotape (Panasonic MD-830AG). Endothelial-independent vasodilatation is induced after sublingual administration of a 400-µg metered dose of NTG (Solinitrina spray; Almirall Prodesfarma, Barcelona, Spain) and is expressed as the percentage of change in the arterial diameter 3 min later. A scan was recorded from 2 min after NTG administration during 70 s. All images registered on super-VHS tape were analyzed afterward by two independent observers blinded to the randomization of the subject and the stage of the experiment. Each observer analyzed the arterial diameter for four cardiac cycles for each condition, and these measurements were averaged. Previous to the initiation of the study in diabetic subjects, validation of this technique was performed through the evaluation of reproducibility inter- and intraobserver in 22 healthy subjects {12 men and 10 women, mean age 30.1 yr [95% confidence interval (CI) 27.1, 33.2], BMI = 22.6 kg/m2 (CI 21.3, 23.8)}. Measurements were performed by two observers (A and B). The intraclass correlation coefficient of fixed effects between observers A and B was 0.90. The coefficient of variation (CV) between means obtained by observers A and B was 9%. The CV obtained by a same observer was 3%. The reproducibility (CI 95%) was 0.27 mm (observer A). With observer B the CV was 4%, with a reproducibility (CI 95%) of 0.39 mm. The study of the variability of the means by the same observer in five consecutive days showed a CV of 6% (observer A) and 2% (observer B). The NTG-induced vasodilation correlated with basal artery diameter (r = -0.67; P = 0.025).

Analytical methods. Serum creatinine was determined by a routine laboratory method. HbA1c was measured by high-performance liquid chromatography with the use of a fully automated glycosylated hemoglobin analyzer system (Hitachi L-9100; reference level 4.6-5.43%).

Plasma sTNFR1 and sTNFR2 levels. Plasma samples were obtained at baseline and were stored at -30°C until assay. Plasma sTNFR1 and sTNFR2 levels were analyzed by commercially available solid-phase enzyme-amplified sensitivity immunoassays (EASIA) MEDGENIX sTNFR1 and sTNFR2 EASIA (BioSource Europe, Fleunes, Belgium). The intra- and interassay CV were <7 and 9%, respectively. sTNFR1 EASIA does not cross-react with sTNFR2. TNF-alpha does not interfere with the assay.

Measurement of TNFR1 expression levels in monocytes by flow cytometry. Blood cells sedimented through Ficoll-Hypaque gradients were washed with RPMI 1640 medium, and monocytes [identified by staining with anti-CD14 monoclonal antibodies (Becton-Dickinson)] were analyzed for CD120a (TNFR1) expression by flow cytometry using biotinylated anti-CD120a monoclonal antibody (Caltag Laboratories, Burlingame, CA) and avidin-phycoerythrin (Southern Biotechnology Associated). Cells (4 × 105) were incubated with conjugated antibodies or isotype controls for 30 min at 4°C, washed two times with PBS containing 0.5% BSA, and incubated with avidin-phycoerythrin for 30 min at 4°C. The cells were washed again, resuspended in 500 µl of PBS, and analyzed on a FACS Calibur flow cytometer (Becton-Dickinson).

Study of shedding. Blood cells sedimented through Ficoll-Hypaque gradients were washed, resuspended at 2 × 106 cells/ml, and incubated at 37°C for 15 or 30 min in RPMI 1640 medium containing 1% FCS with or without phorbol 12-myristate 13-acetate (PMA, 10 ng/ml; Sigma). Blood cells sedimented through Ficoll-Hypaque gradients were washed, resuspended at 2 × 106 cells/ml, and incubated at 37°C for 15 or 30 min in RPMI 1640 medium containing 1% FCS with or without PMA. Monocytes were analyzed for CD120a and CD120b expression before and after PMA activation for 15 and 30 min (Fig. 1). L-selectin expression was measured as an internal control. We calculated the percentage of induced shedding (SHED) by
%SHED R1<IT>t</IT><SUB>15</SUB> = 100 <FENCE>1 − <FR><NU>PMA<IT>t</IT>15R1 − NC PMA<IT>t</IT>15R1</NU><DE>⊘ <IT>t</IT>15R1 − NC ⊘ <IT>t</IT>15R1</DE></FR></FENCE>

%SHED R1<IT>t</IT><SUB>30</SUB> = 100 <FENCE>1 − <FR><NU>PMA<IT>t</IT>30R1 − NC PMA<IT>t</IT>30R1</NU><DE>⊘ <IT>t</IT>30R1 − NC ⊘ <IT>t</IT>30R1</DE></FR></FENCE>

%SHED R2<IT>t</IT><SUB>15</SUB> = 100 <FENCE>1 − <FR><NU>PMA<IT>t</IT>15R2 − NC PMA<IT>t</IT>15R2</NU><DE>⊘ <IT>t</IT>15R2 − NC ⊘ <IT>t</IT>15R2</DE></FR></FENCE>

%SHED R2<IT>t</IT><SUB>30</SUB> = 100 <FENCE>1 − <FR><NU>PMA<IT>t</IT>30R2 − NC PMA<IT>t</IT>30R2</NU><DE>⊘ <IT>t</IT>30R2 − NC ⊘ <IT>t</IT>30R2</DE></FR></FENCE>

%SHED LSEL<IT>t</IT><SUB>30</SUB>

 = 100 <FENCE>1 − <FR><NU>PMA<IT>t</IT>30LSEL − NC PMA<IT>t</IT>30LSEL)</NU><DE>⊘ <IT>t</IT>30LSEL − NC ⊘ <IT>t</IT>30R2</DE></FR></FENCE>
where PMAt15R1, PMAt30R1, PMAt15R2, and PMAt30R2 are the mean fluorescence intensities of TNFR1 and TNFR2 in monocytes incubated at 37°C with PMA for 15 and 30 min; NC PMAt15R1, NC PMAt15R2, NC PMAt30R1, and NC PMAt30R2 are the mean fluorescence intensities of negative controls for these conditions; oslash  t15R1 and oslash  t30R1, oslash  t15R2, and oslash  t30R2 are the mean fluorescence intensities of TNFR1 or TNFR2 in monocytes incubated at 37°C without PMA for 15 and 30 min; NC oslash  t15R1, NC oslash  t15R2, NC oslash  t30R1, and NC oslash  t30R2 are the mean fluorescence intensities of negative controls for these conditions; and LSEL and *t30LSEL are the mean fluorescence intensities of L-selectin incubated without PMA at 0 and 30 min and PMAt30LSEL with PMA for 30 min.


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Fig. 1.   Study of flow cytometry to evaluate shedding of tumor necrosis factor-alpha (TNF-alpha ) receptors using a biotinylated antihuman TNF-alpha receptor 1 (TNFR1) antibody and avidin-phycoerythrin. Decrease of cell surface TNFR1 expression in monocytes incubated with or without phorbol 12-myristate 13-acetate (PMA) for 30 min.

Serum nitrate/nitrite levels. Serum nitrate/nitrite levels were measured with the Nitric Oxide Colorimetric Assay Kit (Calbiochem-Novabiochem, San Diego, CA), based on the Griess method. Amounts of nitrite in the plasma were estimated by a standard curve obtained from enzymatic conversion of KNO3 to nitrite. The total (pro and active) matrix metalloproteinase-9 (MMP-9) in serum samples was measured using a commercially available enzyme-linked immunosorbent assay kit [Quantikine Human MMP-9 (total) Immunoassay; R&D Systems, Minneapolis, MN] according to the manufacturer's instructions.

Exercise Test: Study Protocol

Twenty diabetic patients (11 with DM-1 and 9 with DM-2) were allocated to a training program lasting 3 mo at the same fitness center in all cases. The protocol and blood pressure changes in these patients have been published previously (33). In brief, patients were instructed to attend the fitness center at least 3 times/wk and did not modify their usual daily activities. An individualized aerobic exercise program was designed according to the patients' characteristics and degree of physical fitness, which was determined by means of a treadmill exercise test carried out at the beginning of the study and repeated after 3 mo of training. All exercise sessions were supervised by a coach specifically trained by the investigators for that purpose. Each session included 10 min of warm-up, 30-40 min of aerobic activity, and 10 min of cool down. The exercise consisted mainly of walking or running on a treadmill, cycling, or a combination of both. Initially, subjects exercised at an intensity corresponding to 60-65% maximal O2 uptake (VO2 max; 1-2 wk) and 65-75% VO2 max thereafter to improve their aerobic capacity. All patients had been instructed in diabetes care (diet, blood glucose monitoring, insulin administration, self-adjustment, etc.) and presented stable body weight and glycemic control.

Patients were seen in the outpatient unit every 4 wk to reinforce their program compliance and were instructed on insulin dose modifications. Aerobic capacity, anthropometric parameters, ambulatory blood pressure, glycemic profile, and laboratory analyses were evaluated before and after the planned exercise program. Twenty-four-hour ambulatory blood pressure was registered using a Spacelab 90207 device (Spacelab, Redmond, WA) both before and after the exercise program. During the estimated waking hours (0700-2300), blood pressure was recorded every 20 min and every 30 min during the overnight period.

Statistical Methods

We used a chi 2 test for comparisons of proportions and unpaired or paired t-tests for comparisons of quantitative variables. Before statistical analysis, normal distribution and homogeneity of the variances were tested. Parameters that did not fulfill these tests were log transformed. The relations between variables were analyzed by simple correlation, partial correlation, and multiple regression in a stepwise manner. Levels of statistical significance were set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General characteristics of the study subjects are shown in Table 1 (means ± SD). Age, years of evolution of diabetes, glycosylated hemoglobin, or renal function did not influence the R2/R1 in the diabetic groups. In contrast, a significant negative association between R2/R1 and serum creatinine was observed in the control group (r = -0.17, P = 0.01). R2/R1 was significantly higher in DM-2 than in the other groups (Table 1). The differences in R2/R1 paralleled differences in mean SBP and DBP among groups (Table 1). These differences in R2/R1 were mainly because of decreased circulating sTNFR1 levels in DM-2 compared with age- and BMI-matched control subjects (1.60 ± 0.8 vs. 2.14 ± 0.5 ng/ml, P = 0.001), whereas sTNFR1 levels were similar in DM-1 patients to those present in age- and BMI-matched control subjects (2.10 ± 0.6 vs. 1.97 ± 0.5 ng/ml, P = 0.2). The type of treatment of diabetes or associated drugs was not associated with R2/R1 differences.

                              
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Table 1.   Anthropometrical and clinical variables in the study subjects

Insulin Sensitivity, Vascular Dysfunction, and Circulating sTNFR1 and sTNFR2 Concentrations

In a subsample of 19 consecutive unselected DM-2 patients, we evaluated insulin sensitivity in relation to TNFR1 and TNFR2 shedding. These patients were not significantly different in age, years of diabetes, or usual treatment compared with the remaining group of DM-2 patients. Insulin sensitivity was negatively associated with serum sTNFR2 levels (r = -0.66, P = 0.002), as previously found in nondiabetic subjects (16). Insulin sensitivity appeared to be associated with serum sTNFR1 (r = -0.49, P = 0.03), but the latter association disappeared after one subject with sTNFR1 >3.5 ng/ml was excluded.

The mean ± SD vasodilatory response to glyceryl trinitrate was 14 ± 7% (range 2.3-27.6%). This response was positively associated with serum sTNFR1 concentration (r = 0.62, P = 0.01, n = 16; Fig. 2), indicating that the lower the sTNFR1, the lower the vasodilation. Serum nitrate/nitrite concentrations were used as a surrogate of NO action, i.e., the higher the levels, the higher the resistance to NO action. Serum nitrate/nitrite concentrations correlated positively with DBP (r = 0.58, P = 0.02).


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Fig. 2.   Study of the association between the vasodilatory response to glyceryl trinitrate and serum-soluble TNF-alpha receptor 1 (sTNFR1) concentration.

Insulin Sensitivity and In Vitro Shedding of TNFR1 and TNFR2

The TNFR1 density in peripheral blood monocytes was similar in DM-2 patients compared with five nondiabetic subjects (mean ± SE, 47.8 ± 22 vs. 38.7 ± 8.5, P = not significant). After PMA, shedding of TNFR1 was significantly decreased in DM-2 compared with control subjects (35.6 ± 9.8 vs. 65.1 ± 19.2, P = 0.04). In vitro TNFR1 shedding was positively associated with insulin sensitivity in DM-2 patients (r = 0.54, P = 0.04, n = 14; Fig. 3). Conversely, TNFR2 shedding was negatively associated with insulin sensitivity (r = -0.54, P = 0.03). The shedding of L-selectin, used as control, showed no significant association with insulin sensitivity.


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Fig. 3.   Linear correlation of the association between the change in shedding after PMA and insulin sensitivity in type 2 diabetic patients. DM-2, patients with type 2 diabetes; sTNFR2, soluble TNF-alpha receptor 2.

sTNFR1 and sTNFR2 Changes After Lowering of Blood Pressure

In a sample of 20 eligible patients, previously reported (33), we evaluated whether changes in blood pressure run in parallel with variations in R2/R1. The main features of these patients were as follows: 11 type 1 diabetic patients (6 men, 5 women; mean age 26.4 ± 6.5; range: 19-42 yr) with a mean diabetes duration of 6 ± 5 (0.7-16.2) yr and 9 type 2 diabetic patients (7 men, 2 postmenopausal women; mean age: 54 ± 5.2; range: 48-64 yr) with a mean diabetes duration of 8.2 ± 5 (2.1-19.8) yr. All were treated with diet plus insulin except two type 2 diabetic patients who were on diet therapy alone. Ten patients smoked, and six were regular alcohol consumers (<200 g/wk). Two patients had background retinopathy and two microalbuminuria.

At baseline, R2/R1 correlated negatively with VO2 max in type 2 diabetic patients (r = -0.77, P = 0.003). This association was not statistically significant in type 1 diabetic patients. After the exercise program, we observed a positive association between the decrease in mean blood pressure during the 24-h ambulatory blood pressure recording and the decrease in R2/R1 (r = 0.46, P = 0.042 for SBP; r = 0.47, P = 0.03 for DBP, and r = 0.45, P = 0.04 for mean blood pressure; Fig. 4). When the analysis was performed separately in DM-1 and DM-2 groups, this association remained significant only in the DM-2 group (r = 0.71, P = 0.032; Fig. 4).


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Fig. 4.   Linear correlation of the association between the change in the sTNFR2-to-sTNFR1 ratio and the change in mean systolic blood pressure during a 24-h ambulatory blood pressure recording in diabetic patients (open circle  and , type 1 and type 2 diabetic patients, respectively).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Abnormalities in immune system function and inflammatory mediators have been claimed to be responsible for the onset of hypertension (15, 18) and type 2 diabetes (17). Type 2 diabetes mellitus and insulin resistance have been hypothesized as chronic inflammatory disorders (17, 31). Here we report that insulin resistance was associated with increased serum sTNFR2 concentration and decreased TNFR1 shedding in type 2 diabetic patients. We also found that altered shedding of TNF-alpha receptors is linked to vascular dysfunction and blood pressure. After blood pressure is decreased, circulating sTNFR1 increases and sTNFR2 decreases, leading to a lower R2/R1. We propose that the latter is mediated through increased and decreased shedding, respectively, of TNFR1 and TNFR2.

Interestingly, genetic alterations in TNFR1 shedding have been linked to autoinflammatory syndromes (20). Stimulation with PMA, which induces metalloprotease-mediated cleavage of TNFR1, resulted in much less clearance of membrane receptor in patients with the autoinflammatory syndrome than in controls, leading to decreased sTNFR1 levels (20). Thus DM-2 and autoinflammatory syndromes share a low circulating sTNFR1 concentration. Sequencing of the TNFR1 cDNA in some of our DM-2 patients showed that they did not carry this mutation (Fernandez-Real, Vendrell, and Ricart, unpublished results).

Endogenous sTNFR1 is an excellent marker of inflammation (26). In animal models of inflammation and autoimmunity, administration of recombinant sTNFR1 arrests collagen arthritis and protects against lethal endotoxemia (21). In humans, treatment of septic shock patients with p55 TNF fusion protein, but not with p75 TNF fusion protein or antibodies (anti-TNF), reduced the mortality rate (1). Serum sTNFR1 concentrations in DM-1 patients were similar to those present in age- and BMI-matched control subjects, but the sTNFR1 concentration was decreased significantly in DM-2 patients. The circulating density of TNFR1 on the membrane of circulating monocytes and lymphocytes was similar in DM-2 patients and in controls. After PMA stimulation, however, a decreased shedding of TNFR1 was evident in DM-2 subjects, and this shedding was associated with insulin resistance. Decreased shedding was specific for TNFR1 in contrast to TNFR2 and L-selectin. Recent studies have shown that different mechanisms are involved in shedding of both TNF-alpha receptors as follows: a membrane-bound nonmatrix metalloproteinase is involved in TNFR1 shedding, and a serine proteinase contributes to TNFR2 shedding (14). The TNFR1 stimulation with excess TNF-alpha seemed to transactivate preferential TNFR2 shedding (4).

We had previously demonstrated that exercise led to decreased blood pressure in DM-2 subjects (33). In fact, physical exercise is a well-established method to enhance insulin sensitivity, making it a useful approach for the treatment of diabetes (6). We evaluated R2/R1 before and after a training program that lasted 3 mo. At baseline, the R2/R1 was significantly associated with VO2 max, an excellent predictor of insulin sensitivity (10, 23), in DM-2 patients. This finding confirms R2/R1 as a good correlate of insulin action. R2/R1 decreased after the training program, and the change in R2/R1 was significantly associated with the decrease in mean blood pressure during a 24-h recording. This association was only observed in DM-2 patients. Thus changes in R2/R1 run in parallel with blood pressure variations in DM-2 and possibly with insulin sensitivity.

In recent studies, insulin resistance has been found to be associated with an impairment in the ability of NO to generate its messenger (cGMP), leading to an increase in nitrate/nitrite (8, 29, 30, 32). This increase could represent an effort to compensate for the defect in cGMP production (30). We found that the vasodilation in response to glyceryl trinitrate, a nitric oxide donor, was associated with serum sTNFR1 concentration: the lower the sTNFR1 concentration, the lower the vasodilation. Because NO was recently demonstrated to enhance TNFR1 shedding (25), these findings merit further research.

From our findings, we suggest that the resistance to NO action in DM-2 patients may lead to decreased vasodilatory capacity, decreased TNFR1 shedding, and a subsequent increased R2/R1. The latter would potentiate the effects of TNF-alpha through an increased plasma half-life (4), finally leading to hypertension. Exercise would enhance NO action (19a), resulting in vasodilation, increased TNFR1 shedding, decreased R2/R1, and lowering of blood pressure. Our scheme is summarized in Fig. 5.


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Fig. 5.   Approach to the physiopathology of the hypothesized relationship among blood pressure, shedding of TNFR1, sTNFR2/ sTNFR1, and nitric oxide (NO) and insulin actions.

Some authors have found that decreased shedding of TNF-alpha receptors was mostly related to the development of disease than to elevated secretion of TNF-alpha itself (28). This suggests that the response of cells to TNF-alpha can be regulated by the number of remaining functional receptors on the cell surface, and this number will be greater if shedding is reduced. Thus the relatively high levels of unshed, functionally intact TNFR1 remaining on the cell surface may lead to greater responsiveness to TNF-alpha at physiological concentrations, resulting in increased sensitivity of the TNF-alpha -TNFR1 axis to TNF-alpha . Furthermore, sustained upregulation of human TNFR2 production in transgenic mice leads not only to an upregulated level of shed soluble receptors but also to a chronic accumulation of the receptor on the cell surface (13). All of these events would provide DM-2 patients with the ability to be hyperresponders to circulating TNF-alpha . In fact, DM-2 subjects showed an increased R2/R1 as a probable reflection of TNF-alpha action. To further test this hypothesis, we also measured in these same patients the plasma concentration of MMP-9. We found a positive relationship between the density of TNFR1 in circulating monocytes and circulating MMP-9 (r = 0.56, P = 0.01; data not shown). Because MMP-9 synthesis and secretion are under the control of TNF-alpha (35), these findings favor the hypothesis of increased sensitivity to TNF-alpha in the presence of unshed TNFR1.

In summary, our findings suggest that insulin resistance and hypertension are linked to altered TNFR1 and TNFR2 shedding, leading to increased R2/R1 in DM-2 patients. An increased R2/R1 lowers after decreasing blood pressure, which indicates that it is reversible and not genetically determined. Whether alterations in shedding of peripheral white blood cells is also found in another cell type remains to be established.


    FOOTNOTES

Address for reprint requests and other correspondence: J. M. Fernandez-Real, Unitat de Diabetologia, Endocrinologia i Nutricio, Univ. Hospital of Girona "Dr Josep Trueta," Carretera de França s/n, 17007 Girona, Spain (E-mail: endocrino{at}htrueta.scs.es).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 13, 2001;10.1152/ajpendo.00444.2001

Received 3 October 2001; accepted in final form 7 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, E, Glauser MP, Butler T, Garbino J, Gelmont D, Laterre PF, Kudsk K, Bruining HA, Otto C, Tobin E, Zwingelstein C, Lesslauer W, and Leighton A. p55 Tumor necrosis factor receptor fusion protein in the treatment of patients with severe sepsis and septic shock. A randomized controlled multicenter trial Ro 45-2081 study group. JAMA 277: 1531-1538, 1997[Abstract].

2.   Aderka, D, Engelmann H, Maor Y, Brakebusch C, and Wallach D. Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors. J Exp Med 175: 323-329, 1992[Abstract].

3.   Aderka, D, Engelmann H, Shemer-Avni Y, Hornik V, Galil A, Sarov B, and Wallach D. Variation in serum levels of the soluble TNF receptors among healthy individuals. Lymphokine Cytokine Res 11: 157-159, 1992[ISI][Medline].

4.   Aderka, D, Sorkine P, Abu-Abid S, Lev D, Setton A, Cope AP, Wallach D, and Klausner J. Shedding kinetics of soluble Tumor necrosis factor (TNF) receptors after systemic TNF leaking during isolated limb perfusion. Relevance to the pathophysiology of septic shock. J Clin Invest 101: 650-659, 1998[Abstract/Free Full Text].

5.   Adler, AI, Stratton IM, Neil HA, Yudkin JS, Matthews DR, Cull CA, Wright AD, Turner RC, and Holman RR. Association of systolic blood pressure with macrovascular, and microvascualr complications of type 2 diabetes (U.KPDS 36): prospective observational study. Br Med J 321: 412-419, 2000[Abstract/Free Full Text].

6.   Albright, A, Franz M, Hornsby G, Kriska A, Marrero D, Ullrich I, and Verity LS. American College of Sports Medicine position stand. Exercise and type 2 diabetes. Med Sci Sports Exerc 32: 1345-1360, 2000[ISI][Medline].

7.   Brasier, AR, Li J, and Wimbish KA. Tumor necrosis factor activates angiotensinogen gene expression by the Rel A. transactivator. Hypertension 27: 1009-1017, 1996[Abstract/Free Full Text].

8.   Catalano, M, Carzaniga G, Perilli E, Jun T, Scandale G, Andreoni S, and Carotta M. Basal nitric oxide production is not reduced in patients with noninsulin-dependent diabetes mellitus. Vasc Med 2: 302-305, 1997[Medline].

9.   Celermajer, DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, and Deanfield JE. Non-invasive detection of endothelial dysfunction in children, and adults at risk of atherosclerosis. Lancet 340: 1111-1115, 1992[ISI][Medline].

10.   Clausen, JO, Borch-Johnsen K, Ibsen H, Bergman RN, Hougaard P, Winther K, and Pedersen O. Insulin sensitivity index, acute insulin response, and glucose effectiveness in a population-based sample of 380 young healthy Caucasians. Analysis of the impact of gender, body fat, physical fitness, and life-style factors. J Clin Invest 98: 1195-1209, 1996[Abstract/Free Full Text].

11.   Cseh, K, Winkler Z, Melczer Z, and Baranyi E. The role of tumour necrosis factor (TNF)-alpha resistance in obesity, and insulin resistance. Diabetologia 43: 525, 2000[ISI][Medline].

12.   Dörffel, Y, Lätsch C, Stuhlmüller B, Schreiber S, Scholze S, Burmester GR, and Scholze J. Preactivated peripheral blood monocytes in patients with essential hypertension. Hypertension 34: 113-117, 1999[Abstract/Free Full Text].

13.   Douni, E, and Kollias G. A critical role of the p75 tumor necrosis factor receptor (p75TNFR.) in organ inflammation independent of TNF, lymphotoxin alpha , or the p55TNFR. J Exp Med 188: 1343-1352, 1998[Abstract/Free Full Text].

14.   Dri, P, Gasparini C, Menegazzi R, Cramer R, Alberi L, Presani G, Garbisa S, and Patriarca P. TNF induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor, and involvement of a membrane-bound and non-matrix metalloproteinase. J Immunol 165: 2165-2172, 2000[Abstract/Free Full Text].

15.   Dzielak, DJ. The immune system and hypertension. Hypertension 19, Suppl1: I36-I44, 1992[ISI][Medline].

16.   Fernández-Real, JM, Broch M, Ricart W, Gutiérrez C, Casamitjana R, Vendrell J, and Richart C. Plasma levels of the soluble fraction of Tumor Necrosis Factor Receptor-2, and insulin resistance. Diabetes 47: 1757-1762, 1998[Abstract].

17.   Fernández-Real, JM, and Ricart W. Insulin resistance, and inflammation in an evolutionary perspective. The contribution of cytokine genotype/phenotype to thriftiness. Diabetologia 42: 1367-1374, 1999[ISI][Medline].

18.   Fu, MLX Do immune system changes have a role in hypertension? J Hypertens 13: 1259-1265, 1995[ISI][Medline].

19.   Higashi, Y, Sasaki S, Kurisu S, Yoshimizu A, Sasaki N, Matsuura H, Kajiyama G, and Oshima T. Regular aerobic exercise augments endothelium-dependent vascular relaxation in normotensive as well as hypertensive subjects: role of endothelium-derived nitric oxide. Circulation 100: 1194-1202, 1999[Abstract/Free Full Text].

19a.   Kahaleh, MB, and Fan PS. Effect of cytokines on the production of endothelin by endothelial cells. Clin Exp Rheumatol 15: 163-167, 1997[ISI][Medline].

20.   McDermott, MF, Aksentijevich I, Galon J, McDermott E, Ogunkolade BW, Centola M, Mansfield E, Gadina M, Karenko L, Pettersson T, McCarthy J, Frucht DM, Aringer M, Torosyan Y, Teppo AM, Wilson M, Karaarslan HM, Wan Y, Todd I, Wood G, Schlimgen R, Kumarajeewa TR, Cooper SM, Vella JP, Amos CI, Mulley J, Quane KA, Molloy MG, Ranki A, Powell RJ, Hitman GA, O'Shea J, and Kastner DL. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 97: 133-144, 1999[ISI][Medline].

21.   Mohler, KM, Torrance DS, Smith CA, Goodwin RG, Stremler KE, Fung VP, Madani H, and Widmer MB. Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia, and function simultaneously as both TNF carriers and TNF antagonists. J Immunol 151: 1548-1561, 1993[Abstract/Free Full Text].

22.   Nophar, Y, Kemper O, Brakebusch C, Engelmann H, Zwang R, Aderka D, Holtmann H, and Wallach D. Soluble forms of tumor necrosis factors (TNFRs) The cDNA for the type I TNFR, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor. EMBO J 9: 3269-3278, 1990[Abstract].

23.   Nuutila, P, Knuuti MJ, Maki M, Laine H, Ruotsalainen U, Teras M, Haaparanta M, Solin O, and Yki-Jarvinen H. Gender, and insulin sensitivity in the heart and in skeletal muscles. Studies using positron emission tomography. Diabetes 44: 31-36, 1995[Abstract].

24.   Nyui, N, Tamura K, Yamaguchi S, Nakamaru M, Ishigami T, Yabana M, Kihara M, Ochiai H, Miyazaki N, Umemura S, and Ishii M. Tissue angiotensinogen gene expression induced by lipopolysaccharide in hypertensive rats. Hypertension 30: 859-867, 1997[Abstract/Free Full Text].

25.   Okuyama, M, Yamaguchi S, Yamaoka M, Nitobe J, Fujii S, Yoshimura T, and Tomoike H. Nitric oxide enhances expression, and shedding of tumor necrosis factor receptor I (p55) in endothelial cells. Arterioscler Thromb Vasc Biol 20: 1506-1511, 2000[Abstract/Free Full Text].

26.   Olsson, I, Gatanaga T, Gullberg U, Lantz M, and Granger GA. Tumour necrosis factor (TNF) binding proteins (soluble TNF receptor forms) with possible roles in inflammation, and malignancy. Eur Cytokine Netw 4: 169-180, 1993[ISI][Medline].

27.   Pausova, Z, Deslauriers B, Gaudet D, Tremblay J, Kotchen TA, Larochelle P, Cowley AW, and Hamet P. Role of tumor necrosis factor-alpha gene locus in obesity, and obesity-associated hypertension in French Canadians. Hypertension 36: 14-19, 2000[Abstract/Free Full Text].

28.   Pellegrini, JD, Puyana JC, Lapchak PH, Kodys K, and Miller GC. Membrane TNFalpha/TNFR A ratio correlates to MODS score and mortality. Shock 6: 396, 1996.

29.   Piatti, PM, Fragasso G, Monti LD, Caumo A, Van Phan C, Valsecchi G, Costa S, Fochesato E, Pozza G, Pontirolli AE, and Chierchia S. Endothelial and metabolic characteristics of patients with angina and angiographically normal coronary arteries. Comparison with subjects with insulin resistance syndrome and normal controls. J Am Coll Cardiol 34: 1452-1460, 1999[ISI][Medline].

30.   Piatti, PM, Monti LD, Zavaroni I, Valsecchi G, Van Phan C, Costa S, Conti M, Sandoli EP, Solerte B, Pozza G, Pontiroli AE, and Reaven G. Alterations in nitric oxide/cyclic-GMP pathway in nodiabetic siblings of patients with type 2 diabetes. J Clin Endocrinol Metab 85: 2416-2420, 2000[Abstract/Free Full Text].

31.   Pickup, JC, and Crook MA. Is type II diabetes mellitus a disease of the innate immune system? Diabetologia 41: 1241-1248, 1998[ISI][Medline].

32.   Pieper, GM. Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension 31: 1047-1060, 1998[Free Full Text].

33.   Rigla, M, Sánchez-Quesada JL, Ordóñez-Llanos J, Prat T, Caixàs A, Jorba O, Serra JR, de Leiva A, and Pérez A. Effect of physical exercise on lipoprotein(a), and low-density lipoprotein modifications in type 1 and type 2 diabetic patients. Metabolism 49: 640-647, 2000[ISI][Medline].

34.   Schroder, J, Stuber F, Gallati H, Schade FU, and Kremer B. Pattern of soluble TNF receptors I, and II in sepsis. Infection 23: 143-148, 1995[ISI][Medline].

35.   Siwik, DA, Chang DLF, and Colucci WS. Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metalloproteinase activity in vitro. Circ Res 86: 1265, 2000.

36.   WHO Expert Committee on Diabetes Mellitus. Second Report, Technical Series. Geneva, Switzerland: World Health Organization, 1980.

37.   Winkler, G, Lakatos P, Salamon F, Nagy Z, Speer G, Kovacs M, Harmos G, Dworaks O, and Cseh K. Elevated serum TNFalpha levels as a link between endothelial dysfunction and insulin resistance in normotensive obese subjects. Diabet Med 16: 207-211, 1999[ISI][Medline].

38.   Zinman, B, Hanley AJG, Harris SB, Kwan J, and Fantus IG. Circulating tumor necrosis factor-alpha concentrations in a native Canadian population with high rates of type 2 diabetes mellitus. J Clin Endocrinol Metab 84: 2172-278, 1999.


Am J Physiol Endocrinol Metab 282(4):E952-E959
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