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Original Studies |
Departments of Clinical and Experimental Medicine (R.B., M.Z., G.D., E.K., A.T., P.T.) and Clinical and Laboratory Medicine (P.C.), University of Padova, 35128 Padova, Italy; and Department of Clinical Medicine and Endocrinology, University of Bari (P.T.), 70124 Bari, Italy
Address all correspondence and requests for reprints to: Prof. P. Tessari, Department of Clinical and Experimental Medicine, Policlinico, Via Giustiniani 2, 35128 Padova, Italy. E-mail: ptessari{at}ux1.unipd.it
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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50%; P < 0.01) and
pool (+40%; P < 0.01) as well as fractional
(+35%; P = 0.08) and absolute (+100%;
P < 0.01) synthetic rates. The plasma glucagon
concentration was positively related (P < 0.005 or
less) to the fibrinogen concentration as well as to fractional and
absolute synthetic rates. Thus, fibrinogen production is markedly
enhanced, and this alteration is likely to determine the observed
hyperfibrinogenemia in type 2 diabetic patients. Hyperglucagonemia may
contribute to the increased fibrinogen production. These findings in
normoalbuminuric patients without clinical complications support the
hypothesis that increased fibrinogen production and plasma
concentrations may precede and possibly contribute to the onset of
clinical cardiovascular complications in type 2 diabetes. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Furthermore, the possible role of increased fibrinogen turnover and concentrations as independent risk factors contributing to cardiovascular disease in type 2 diabetic patients has been difficult to assess because of the confounding effect of preexisting clinical macro- and microvascular complications. Clinical stages of vascular disease are indeed per se associated with increased plasma fibrinogen in nondiabetic patients (5, 13, 14), whereas established macrovascular complications were, in turn, related to higher fibrinogen concentration in a mixed type 1 and type 2 diabetic population (9). Microalbuminuria also represents a sensitive marker of cardiovascular disease (15, 16) that is associated with hyperfibrinogenemia in type 2 diabetes (7, 17). High fibrinogen concentration and turnover observed in advanced nephrotic nondiabetic patients (18) suggest a direct independent relationship between enhanced renal albumin excretion and increased fibrinogen metabolism.
The current study was therefore designed to assess postabsorptive fibrinogen synthesis/secretion in type 2 diabetic patients using precursor-product isotopic relationships with leucine tracer iv infusion (19). Based on a thorough noninvasive screening protocol, all patients were without detectable micro- and macrovascular complications to establish whether possible alterations of fibrinogen turnover may actually precede the onset of clinical micro- and macrovascular disease in type 2 diabetes.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Six male type 2 diabetic patients and seven male age- and weight-matched nondiabetic control subjects were studied. Although invasive angiographic procedures were not considered ethical for the purpose of this protocol, all participants were selected according to strict criteria to exclude the presence of diabetic complications with potential effects on fibrinogen concentration and turnover. Medical history and thorough examination revealed no vascular disease or retinopathy. The presence of microalbuminuria was ruled out based on three negative determinations (<20 µg/min) on 24-h urine collections. Electrocardiogram and carotid artery ultrasound were normal in all patients. As carotid ultrasound lesions are related to the presence of coronary atherosclerosis (20), these negative findings further suggest the absence of ischemic heart disease in the subjects. All participants in the study were also screened with routine tests, which demonstrated normal liver and kidney function. None of the subjects was a current smoker, and only one subject in each group had smoked in the past, quitting more than 5 yr before the study. No patient had clinical signs of intercurrent inflammatory conditions that might directly affect fibrinogen metabolism. The erythrocyte sedimentation rate and leukocyte count were measured on the day before the study and were normal in all subjects.
All patients were treated with oral hypoglycemic agents (four with
sulfonylureas, two with metformin). Two patients and two control
subjects also had mild essential hypertension; the two patients and one
of the nondiabetic subjects were treated with angiotensin-converting
enzyme inhibitors. No other drugs had been taken by any of the
participants for at least 3 months before the study. The mean duration
of diabetes was 11 yr, and metabolic control at the time of the study
was poor, as reflected by the hemoglogin A1c
value (Table 1
).
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The experimental protocol was approved by the ethical committee
of the University of Padova (Padova, Italy), and it complied with the
Helsinki Declaration. The study was carried out according to the
recommendations of the radiation safety officer. Each subject provided
a written consent after the aims and potential risks of the study were
explained to him in detail. Antidiabetic and antihypertensive
medications were withdrawn 24 h before the study. All subjects
were admitted to the Metabolic Unit of the Department of Metabolic
Diseases at 0730 h on the study day at their spontaneous glycemic
levels under postabsorptive conditions after the overnight fast. An
18-gauge polyethylene catheter was placed in an antecubital vein of the
right arm for isotope infusion. A contralateral wrist vein was then
cannulated in a retrograde fashion, and the hand was placed in a box
heated at 55 C for venous-arterialized blood sampling throughout the
study. At 0800 h, a primed (60-fold the constant infusion
rate/min), continuous infusion of the leucine isotope was started.
L-[4,5-3H]leucine
([3H]Leu; 3 µCi/kg BW) was infused in all
the diabetic as well as in four of the control subjects, whereas
L-[1-14C]leucine
([14C]Leu; 1.5 µCi/kg; both isotopes from
Amersham Pharmacia Biotech, Amersham Pharmacia Biotech, Aylesbury, U.K.) was infused in the remaining three
controls. Rates of isotope infusion are reported in Table 3a. Data from
all control subjects were pooled because results in both subgroups were
comparable. Furthermore, based on previous reports, the
[14C]Leu should have led, if anything, to
slightly greater fibrinogen synthesis/secretion rates than the
[3H]leu tracer (21), and this would have, in
turn, determined a small underestimation of the differences between the
two groups without affecting the conclusions of this study (see
Results and Discussion). Blood samples were drawn
every 30 min for 3 h, then every 20 min in the last hour of the
study (i.e. between 180–240 min), for kinetic calculations
under steady state conditions for plasma substrates as well as isotope
specific activities (SA).
Analytical methods
Fibrinogen was isolated from plasma as previously described (19, 22). Shortly, 100 µL of a 1 mol/L CaCl2
solution and 10 IU thrombin in 100 µl deionized water were added to 2
mL plasma to activate the reactions leading to the formation of a
fibrin clot. After 1-h incubation at room temperature the clot was
removed from the sample, gently washed on filtered paper with deionized
water to remove any plasma residues, and hydrolyzed for 48 h at
110 C in 4 mL 4 N HCl. The fibrinogen-derived free amino
acids were then purified through cation exchange AG 50X8 columns (19, 22). The [3H]- or
[14C]Leu SA in the hydrolyzed fibrinogen
samples as well as plasma 
-ketoisocaproate (KIC) SA from either
isotope were measured by high performance liquid chromatography as
previously described (19). The plasma fibrinogen concentration was
determined by nephelometric assay (23). Plasma glucose was measured
using a glucose analyzer 2 (Beckman Coulter, Inc.,
Fullerton, CA). Plasma concentrations of insulin, glucagon, and C
peptide were determined by RIA as previously described (22).
Calculations
Fibrinogen fractional synthesis/secretion rate (FSR; expressed as a percentage of the pool per day-1) was calculated in the last hour of the study under steady state conditions for the plasma precursor pool SA (i.e. [3H]- or [14C]KIC SA). At least four time points were used for the regression analysis of the changes in fibrinogen-bound leucine SA vs. time in each subject. Plasma KIC SA is classically used as index of the intrahepatic precursor pool for fibrinogen synthesis (19, 22, 24). The following standard equation (19, 22) was employed: FSR = [(SAt2 - SAt1)/t2 - t1)/SAprecursor] x 1440 x 100 (Eq I), where SAt2 and SAt1 are fibrinogen-bound leucine specific activities (disintegrations per min/nmol) at time points t2 and t1, respectively; the ratio of their differences represents the slope of their linear regression analysis as previously described. SAprecursor is the plasma KIC SA at steady state, and a factor 1440 is used to express synthesis rates per day (i.e. 24 h or 1440 min). The fibrinogen absolute secretion rate (ASR; expressed as milligrams per day-1) was calculated by multiplying the FSR times the plasma (i.e. intravascular) fibrinogen pool. The intravascular fibrinogen pool was estimated by multiplying plasma fibrinogen concentration (milligrams per L) times the plasma volume (liters), which, in turn, was calculated using standard formula based on body surface (25). Leucine plasma and intracellular rates of appearance, reflecting whole body protein turnover, were calculated by dividing the isotope rate of infusion over the steady state plasma leucine or KIC SA in the last hour of the study (26).
Statistical analysis
Data from the two groups (expressed as the mean ± SE) were compared using the two-tailed Student’s t test for unpaired data. P < 0.05 was considered statistically significant. Plasma triglyceride concentrations in the two groups were compared using the Wilcoxon test because of their nonparametric distribution.
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Results |
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Plasma glucose, insulin, glucagon, and C peptide concentrations
were higher in type 2 diabetic patients than in controls (Table 2). Total and high density lipoprotein
(HDL) cholesterol were comparable in the two groups, whereas plasma
triglyceride concentrations were increased (P < 0.05)
in the patients, as expected (Table 2).
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The steady state leucine and KIC SA were not different between
patients and controls, and no differences were detectable between the
two groups in leucine rates of appearance, reflecting whole body
protein turnover (Table 3).
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The plasma fibrinogen concentration was increased by about 50%
(P = 0.02) in the diabetic compared to the control
subjects (Table 3a), and the plasma fibrinogen pool was also
significantly increased by 60% in the patients (P =
0.01; Table 3a). The slopes of the linear regression analysis between
fibrinogen-bound leucine SA vs. time in the last hour of the
study were steeper in the diabetic than in the control group (Table
3a). The correlation coefficients of linear regression analysis (r)
were 0.95 ± 0.02 and 0.97 ± 0.01 in the diabetic and
control groups, respectively. Fibrinogen FSR was about 35% greater in
the patients than in the control subjects, but this difference did
not reach statistical significance (22.8 ± 3.0% vs.
16.4 ± 0.9%/day; P = 0.08; Fig. 1), whereas
fibrinogen ASR was more than 2-fold greater in the diabetic
patients (2.15 ± 0.36 vs. 1.01 ± 0.07 g/day;
P = 0.01; Fig. 1).
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),
intravascular pool (r = 0.90, P < 0.0001), FSR
(r = 0.69, P < 0.005), and ASR (Fig. 2). The positive correlations between
plasma glucagon and fibrinogen pool, and ASR were significant also in
the diabetic group considered alone (n = 6; P <
0.05; data not shown). No significant correlation (P >
0.1) was found between fibrinogen synthesis/secretion and plasma
glucose concentrations, but a significant correlation was observed
between plasma glucose and fibrinogen concentrations in all subjects
(r = 0.55; P = 0.04).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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All patients in the present report were normoalbuminuric and without detectable micro- and macrovascular complications. The current results therefore also indicate that fibrinogen overproduction as well as hyperfibrinogenemia may precede the onset of clinical vascular complications, which have been previously independently associated with increased fibrinogen concentrations in type 2 diabetes. Although minor preclinical alterations in all subjects cannot be entirely ruled out, they have never been demonstrated to affect fibrinogen concentration per se. Moreover, prospective studies in nondiabetic subjects have shown that plasma fibrinogen retains a strong predictive value for cardiovascular events even at advanced stages of vascular disease (6). Therefore, these data support the hypothesis that increased fibrinogen production and plasma concentration may contribute to the onset of vascular complications and thus represent an independent cardiovascular risk factor in type 2 diabetic patients.
The lack of difference in leucine kinetics between diabetic and control subjects is fully consistent with previous studies (30, 31) and confirms near-normal rates of whole body protein turnover in type 2 diabetes. The simultaneous observation of profound alterations of fibrinogen production conversely indicates that whole body protein turnover may not reflect the differential alterations in turnover rates of specific proteins in different organs and that studies of regional and/or specific protein kinetics are needed in this disease.
Whether the hormonal and metabolic alterations in type 2 diabetes are associated with the observed alteration of fibrinogen production is currently unknown. In this regard the correlation between plasma glucagon and all parameters of fibrinogen metabolism is intriguing. This strong correlation indeed suggests that increased glucagon concentrations (32) may contribute to the increased fibrinogen production and plasma levels in type 2 diabetes. This hypothesis is consistent with our recent report of increased fibrinogen fractional synthetic rate after acute glucagon infusion in healthy humans (33) and with in vitro findings indicating a stimulatory effect of glucagon on the secretion of fibrinogen and other acute phase reactants in cultured hepatocytes (34).
Plasma glucose concentrations were also increased in the patients, as expected. Hyperglycemia has been previously shown to activate the coagulative cascade (35), thus increasing thrombin formation and fibrinogen degradation products, which, in turn, may stimulate hepatic fibrinogen synthesis (36). A positive correlation between plasma glucose and fibrinogen concentration has been reported in large epidemiological studies (8) and was confirmed in the current investigation despite the smaller study population. It is therefore possible that increased plasma glucose contributes to the hyperfibrinogenemia of type 2 diabetes. This hypothesis is consistent with previous findings of less pronounced increments in plasma fibrinogen concentration in type 2 diabetic patients studied under conditions of good metabolic control (10) as well as with the observation of acute reductions of fibrinogen synthesis in type 1 diabetes after normalization of plasma glucose (27). The contribution of increased plasma insulin in the patients to the observed alterations is less straightforward. Available literature data indicate that insulin acutely inhibits fibrinogen production both in type 1 diabetic patients after insulin deprivation (27) and in healthy control subjects (37). Although the effect of insulin on fibrinogen turnover in type 2 diabetes is unknown, an insulin-resistant state also extended to fibrinogen production may be hypothesized in these patients. Alternatively, hyperinsulinemia in diabetic subjects might have blunted an otherwise even higher fibrinogen overproduction.
In the current report the two study groups were matched for body mass index and prevalence of hypertension as well as mean arterial blood pressure. The two groups also had similar and normal total as well as HDL cholesterol levels, whereas plasma triglycerides were increased in the patients, as expected. However, both no relationship and even a negative correlation between plasma fibrinogen and triglyceride concentrations have been previously reported (38). Based on the above observations it is therefore unlikely that these potential confounding factors (38, 39) contributed to the alterations of fibrinogen turnover reported in the current investigation.
In conclusion, we show that fibrinogen production is substantially increased in type 2 diabetic patients, and this alteration is likely to play a key role in the increased fibrinogen concentrations in type 2 diabetes. Increased plasma glucagon concentrations may contribute to the enhanced fibrinogen production. These results in normoalbuminuric patients without detectable vascular complications also indicate that increased fibrinogen production and plasma concentration may precede the onset of clinical cardiovascular disease and might therefore contribute to the increased cardiovascular risk in type 2 diabetes.
Received December 29, 1999.
Revised May 12, 2000.
Accepted May 23, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-ketoisocaproic acid
concentrations and specific activity in plasma and leucine specific
activity in proteins using high-performance liquid chromatography. J Chromatogr. 495:81–94.[Medline]
-ketoisocaproate. Eur J Clin Invest. 23:350–355.[Medline]
-1 acid glycoprotein, -2
(acute phase) globulin and aptoglobin by direct action of hormones on
the isolated perused liver. In: Litwak G, ed. Biochemical actions of
hormones. New York: Academic Press; 159–186.
This article has been cited by other articles:
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  P. Tessari, E. Kiwanuka, R. Millioni, M. Vettore, L. Puricelli, M. Zanetti, A. Gucciardi, M. Tosolini, P. Cogo, V. Carnielli, et al. Albumin and Fibrinogen Synthesis and Insulin Effect in Type 2 Diabetic Patients With Normoalbuminuria Diabetes Care, February 1, 2006; 29(2): 323 - 328. [Abstract] [Full Text] [PDF]
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 R. Barazzoni, E. Kiwanuka, M. Zanetti, M. Cristini, M. Vettore, and P. Tessari Insulin Acutely Increases Fibrinogen Production in Individuals With Type 2 Diabetes but Not in Individuals Without Diabetes Diabetes, July 1, 2003; 52(7): 1851 - 1856. [Abstract] [Full Text] [PDF]
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