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Original Studies |
Unitat de Diabetologia, Endocrinologia i Nutricio, University Hospital of Girona Dr. Josep Trueta, 17007 Girona; and Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona; Hormonal Laboratory, University Hospital Clinic, Barcelona, Spain; and Hospices Civils de Lyon, Laboratoire de la Clinique Endocrinologique, Hôpital de l’Antiquaille, and INSERM U-329, 69321 Lyon Cedex 05, France
Address all correspondence and requests for reprints to: J. M. Fernandez-Real, M.D., Ph.D., Unitat de Diabetologia, Endocrinologia i Nutricio, University Hospital of Girona Dr. Josep Trueta, Carretera de França s/n, 17007 Girona, Spain. E-mail: endocri{at}htrueta.scs.es
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Abstract |
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Because carbohydrate chains influence the biological activity and half-life of glycoproteins, we analyzed the migration profile of CBG by Western blot and the interaction of CBG with lectin, Con A. The results indicated that the CBG mol wt and interaction with Con A did not differ between lean and obese patients. These data favor the hypothesis that the inhibitory effect of insulin on CBG liver secretion might be relevant in vivo and therefore contribute to decrease CBG levels in obese patients with enhanced insulin secretion.
In both men and women, SHBG levels correlated negatively with fasting glucose (r = -0.55; P < 0.0001) and hemoglobin A1c (r = -0.38; P = 0.02) and positively with insulin sensitivity (SI; r = 0.65; P = 0.003 and r = 0.63; P = 0.007 in men and women, respectively), but not with insulin secretion. The disposition index (SI x AIRg) was significantly decreased in the obese, glucose-intolerant subjects, suggesting that AIRg was inadequate for their degree of insulin resistance. The disposition index correlated positively with plasma SHBG levels (r = 0.52; P = 0.001) and negatively with plasma CBG levels (r = -0.54; P = 0.001).
Our data suggest that CBG is a marker of insulin secretion in a similar way as SHBG is a marker of insulin sensitivity. As high plasma CBG levels have been associated with increased incidence of type 2 diabetes, this important issue merits further investigations.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Three groups of subjects were prospectively studied: lean, obese, and obese subjects with glucose intolerance. Inclusion criteria were 1) BMI (weight in kilograms divided by the square of height in meters) greater than 30 and less than 40 kg/m2 for obese subjects and less than 27 (men) or less than 25 kg/m2 (women) for lean subjects, 2) the absence of any systemic disease, and 3) the absence of any infections in the previous month. None of the subjects was taking any medication (including glucocorticoids or estrogens) or had any evidence of metabolic disease other than obesity. All subjects reported that their body weight had been stable for at least 3 months before the study; all were normotensive and normolipemic (the latter data are not shown). Liver disease and thyroid dysfunction were specifically excluded by biochemical workup. All women had regular menstrual cycles and were studied on days 3–8 of two consecutive menstrual cycles. The protocol was approved by the hospital ethics committee, and informed consent was obtained from each subject.
Procedures
Each subject’s waist was measured with a soft tape midway between the lowest rib and the iliac crest. The hip circumference was measured at the widest part of the gluteal region. 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. The subjects were required to consume a weight-maintaining diet containing at least 300 g carbohydrate/day and refrained from exertion for 3 days before the study. The subjects also abstained from caffeine and alcohol for 72 h before the tests. An oral glucose tolerance test (OGTT) was performed according to the recommendations of the National Diabetes Data Group (15). After a 12-h overnight fast, glucose was ingested in a dose of 75 g, and blood samples were collected through a venous catheter from an antecubital vein at 0, 30, 60, 90, and 120 min for measurement of serum glucose and insulin. The glucose total area under the curve (AUC glucose) during the OGTT was determined by the trapezoidal method.
Insulin sensitivity was analyzed using the frequently sampled iv glucose tolerance test (FSIGTT) with minimal model analysis as described previously (16, 17). In brief, the experimental protocol started between 0800–0830 h 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 mins, after which glucose (300 mg/kg BW) was injected over 1 min starting at time zero. Additional samples were obtained from a contralateral antecubital vein until 180 min.
The serum glucose level during the FSIGTT was measured in duplicate by the glucose oxidase method with a glucose analyzer 2 (Beckman Coulter, Inc., Brea, CA). The coefficient of variation was 1.9%. The serum insulin level during the FSIGTT was measured in duplicate by monoclonal immunoradiometric assay (Medgenix Diagnostics, Fleunes, Belgium). The lowest limit of detection was 4.0 mU/L. The intraassay coefficient of variation was 5.2% at a concentration of 10 mU/L and 3.4% at a concentration of 130 mU/L. The interassay coefficients of variation were 6.9% and 4.5% at 14 and 89 mU/L, respectively. Blood glycosylated hemoglobin (HbA1c) concentrations were analyzed by high pressure liquid chromatography (Merck Diagnostics), with coefficients of variation below 4%. The range for HbA1c in glucose-tolerant subjects was 3.8–5.43%.
Cortisol, CBG, and SHBG measurements
Serum cortisol was evaluated in samples obtained from the indwelling catheter at -10, 0, 20, and 30 min, respectively, after venepuncture. Its concentration was determined by microparticle enzyme immunoassay (IMX system, Abbott Laboratories, North Chicago, IL), with intra- and interassay coefficients of variation less than 8%, as recently reported (9, 18). The binding capacity of serum CBG was measured in duplicate using a solid phase binding assay (19), and the protein concentration of CBG was measured by RIA as previously described (20). To analyze glycoform variation in CBG, serum samples (50 µL) were subjected to Con A-Sepharose adsorption [300 µL in 50% Tris buffer (50 mmol/L Tris-HCl, pH 7.4; O.5 mol/L NaCl; 1 mmol/L CaCl2; 1 mmol/L MgCl2; and 1 mmol/L MnCl2)]. The concentration of CBG was measured in the supernatant of the Con A-gel for calculating the percentage of CBG that was adsorbed by Con A and refereed as glycosylated CBG. The electrophoretic mobility of CBG was assessed by PAGE (4% stacking and 10% resolving gels) in the presence of SDS (SDS-PAGE), and electroblotting transfer to nitrocellulose membranes were performed according to the method described by Avvakumov and Hammond (21). The sex hormone-binding globulin (SHBG) concentration was measured with a specific immunoradiometric assay for human SHBG (125I-SHBG-Coatria) obtained from BioMérieux eSA (Marcy l’Etoile, France).
Data analysis
Data from the FSIGTT were submitted to computer programs that calculate the characteristic metabolic parameters by fitting glucose and insulin to the minimal model that describes the time courses of glucose and insulin concentrations. The glucose disappearance model, by accounting for the effect of insulin and glucose on glucose disappearance, provides the parameter SI (10-4/min·mU/L), a measure of the effect of insulin concentrations above the basal level to enhance glucose disappearance. The estimation of model parameters was performed according to the MINMOD computer program (22). Insulin secretion from the FSIGTT was calculated as the incremental insulin response from 0–10 min after iv glucose (AIRg). The disposition index (SIAIR) is defined as the product of SI x AIRg. The initial pancreatic insulin response to glucose (IRG) was estimated as the ratio of the incremental area under the insulin curve above the fasting level (I30-I0; milliunits per min/L) to the incremental area under the glucose curve above the fasting level (G30-G0, millimoles per min/L) during the first 30 min of the OGTT and was expressed as: IRG (mU/mmol) = (I30-I0)/(G30-G0).
Statistical analysis
Descriptive results of continuous variables are expressed as the
mean ± SD. Before statistical analysis, normal
distribution and homogeneity of the variables were tested. Parameters
that did not fulfill these tests (CBG, SHBG, AIRg, IRG, SI,
and SIAIR) were log transformed. We used the
2 test for comparisons of proportions. Comparison of
variables across the three groups of subjects was performed by one-way
ANOVA using Fisher’s test for multiple comparisons. Levels of
statistical significance were set at P < 0.05.
Statistical analysis were performed with the BMDP statistical package
(BMDP Statistical software, Cork Technology Park, Cork, Ireland).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. The obese subjects were classified as
glucose tolerant or glucose intolerant according to the criteria of the
National Diabetes Data Group (15). The three groups of subjects were
comparable in sex and age, and the two obese groups were similar in
BMI, waist to hip ratio, and blood pressure (Table 1). Obese intolerant
subjects showed higher fasting and postload glucose levels, higher
fasting insulin levels, and increased HbA1c concentration
than the other groups of subjects (Table 1). Obese subjects displayed
enhanced insulin secretion, after either oral or iv glucose, and a
nonsignificantly different insulin sensitivity index compared to obese
intolerant individuals (Table 1).
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and Fig. 1). When all individuals were analyzed
together, plasma CBG levels positively correlated with fasting glucose
(r = 0.49, P = 0.002), AUC glucose after OGTT
(r = 0.45; P = 0.005; Fig. 2), and HbA1c concentration
(r = 0.35; P = 0.03) and negatively with AIRg
(r = -0.38; P = 0.02; Fig. 2) and IRG (r =
-0.40; P = 0.01), but not with fasting insulin levels
(r = 0.12; P = NS) or insulin sensitivity (r
= -0.11; P = NS; Fig. 3). Those subjects with AIRg
over the median (369.8 mU/L) displayed significantly lower CBG levels
(33.1 ± 4.5 vs. 36.8 ± 5.9 mg/L;
P = 0.04). Nonglycosylated CBG correlated positively
with serum glucose (r = 0.50; P = 0.001) and
negatively with AIRg (r = -0.46; P = 0.004). In
contrast, serum F levels did not correlate with any of these
parameters.
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Mean plasma SHBG levels were significantly (P =
0.002) lower in men (19.4 ± 7.6 nmol/L) than in women (37.1
± 21.2). SHBG levels correlated negatively with fasting glucose
(r = -0.55; P < 0.0001) and HbA1c
(r = -0.38; P = 0.02) and correlated positively
with insulin sensitivity (r = 0.65; P = 0.003 and
r = 0.63; P = 0.007, in men and women,
respectively; Fig. 3), but not with
insulin secretion (r = 0.06; P = NS).
The disposition index (SIAIR), defined as the product of
insulin sensitivity by AIRg, was significantly decreased in the obese
intolerant subjects (Table 1). SIAIR correlated positively
with plasma SHBG levels (r = 0.52; P = 0.001) and
negatively with plasma CBG levels (r = -0.54; P =
0.001).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The negative significant correlation between CBG and the insulin secretion parameters suggests that insulin might decrease CBG liver production and/or increase the CBG plasma clearance rate, or may have both effects. Because carbohydrate chains influence the biological activity and half-life of glycoproteins, we analyzed the migration profile of CBG by Western blot and the interaction of CBG with lectin, Con A. The results indicate that the mol wt of CBG and the interaction with Con A did not differ between lean and obese patients. These data favor the hypothesis that the inhibitory effect of insulin on CBG liver secretion (14) might be relevant in vivo and therefore contribute to the decrease in CBG levels in obese patients with enhanced insulin secretion. From the literature there was previous evidence that nutritional factors influence CBG levels in humans. Indeed, Anderson et al. (23) reported that CBG levels decreased during a high carbohydrate diet compared with those during a high protein diet. Because a high carbohydrate diet normally induces an increased insulinemic response, these results support the idea that insulin could be a inhibitory mechanism for circulating CBG. However, the finding of decreased CBG levels in obese patients with enhanced insulin secretion is in apparent contrast with the normal CBG levels that have been reported in women with anorexia nervosa (24). Because anorectic patients have extremely low insulin levels (25), the inhibitory effect of insulin on CBG could indicate hepatic insulinization when insulin secretion is normal or enhanced, but not in the circumstance of insulin deprivation, as in glucose intolerance, diabetes, or anorexia nervosa. With this aim, our obese glucose-intolerant subjects had impaired insulin secretion together with a decreased disposition index (SIAIR), suggesting that insulin secretion was inadequate for their degree of insulin resistance. This could explain why low CBG levels were restricted to obese subjects with enhanced insulin secretion. On the other hand, the relationship between hepatic insulinization and CBG levels, is in agreement with the report that CBG levels are increased in type 1 pubertal diabetics (26).
In this report we have only investigated the effects of carbohydrate metabolism on CBG. Given the complex and different mechanisms involved in the total control of CBG, we contribute to characterize some aspects of this regulation in a somewhat limited perspective. On the other hand, the decrease in CBG has been related at least in part to enhanced interleukin-6 (IL-6) secretion (27). In a recent study, IL-6 infusion was found to decrease CBG levels in healthy volunteers (28). In this sense, increased production of IL-6 by sc adipose tissue has been described in man, and the arterial plasma concentration of IL-6 was found to be proportional to body mass index and percent body fat (29), thus suggesting an additional possible mechanism by which plasma CBG would be decreased in obese subjects with preserved insulin secretion.
Interestingly, one epidemiological study has shown that high plasma CBG is associated with increased incidence of type 2 diabetes (30). In this study, the increased incidence of diabetes was also confined to the lowest quintile of SHBG values (30). SHBG has been generally found to be negatively correlated with BMI and fasting insulin levels (31). Whether SHBG is a marker of insulin secretion and/or insulin resistance has been debated. Yki-Järvinen et al. (32) reported that serum SHBG levels were disproportionately increased in type 1 diabetic patients when related to insulin sensitivity, but were appropriate when related to estimated portal insulin concentrations. The researchers suggested that SHBG is a useful marker of insulin sensitivity only in individuals with intact insulin secretion. In a study of type 2 diabetic men, Katsuki et al. (33) also reported that SHBG was an index of insulin resistance only in the hyperinsulinemic state. Our data showing that SHBG levels, independently of insulin secretion, correlated positively with insulin sensitivity are in agreement with these two studies.
It could be argued that insulin sensitivity is likely to control the baseline fasting insulin levels and probably is the dominate influence on the overall insulin secretion over 24 h. In vitro data suggest that the inhibitory effect of insulin on CBG production is more pronounced than the inhibitory effect of insulin on SHBG production by Hep G2 cells (14). This might explain why CBG is more strongly associated with insulin secretion than SHBG. In addition to CBG, the plasma concentration of another binding proteins seem to be best explained by insulin secretion or portal insulin. In type 1 diabetes mellitus, the reductions observed in specific GH-binding protein (GHBP) and insulin-like growth factor-binding protein-3 are not normalized after intensified sc insulin therapy. The attainment of normal levels of these binding proteins obtained with ip insulin therapy (which allows primary portal insulin absorption) provides direct evidence of the central role of portal insulin in the regulation of this system (34). Similar events might be hypothesized for CBG.
In summary, our results suggest that plasma CBG levels vary according to insulin secretion. Whether high CBG levels are simply markers of low insulin secretion or determine subtle metabolic changes awaits further investigation.
Received March 17, 1999.
Revised May 17, 1999.
Accepted May 19, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene Ncol polymorphism influences the
relationship among insulin resistance, percent body fat and increased
serum leptin levels. Diabetes. 46:1468–1472.[Abstract]
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