Plasma Levels of Tumor Necrosis Factor-α, Angiotensin II, Growth Hormone, and IGF-I Are Not Elevated in Insulin-Resistant Obese Individuals With Impaired Glucose Tolerance

We studied 100 Caucasian men and women aged 25-51 years. Each proband was subjected to a 75-g oral glucose tolerance test (OGTT) and a hyperinsulinemic-euglycemic clamp. The subjects were divided into two groups on the basis of the euglycemic clamp results. The threshold amount of glucose infused in the euglycemic clamp for insulin resistance was <40 μmol · kg^-1 ·min^-1 and for insulin sensitivity >50 μmol ·kg^-1 · min^-1(20). All subjects were chosen according to the following inclusion criteria:

• age between 25 and 55 years

• no history of any metabolic disorder

• GAD determined by an radioimmunoassay (RIA) (Brahms, Berlin) and/or islet cell antibody determined by an immuno-histochemical method undetectable

• no dyslipidemia

• no thyroid dysfunction

• no concomitant medication

• no regular alcohol or drug consumption

In addition to these criteria, the IR group (48 subjects with IGT and insulin resistance) was defined as follows:

• fasting plasma glucose <6.0 mmol/l(24), 120-min plasma glucose after 75-g oral glucose load <11.1 mmol/l (no diagnosis of type 1 or type 2 diabetes)

• positive family history of type 2 diabetes or gestational diabetes

The IR group was recruited from 214 subjects who were screened by OGTTs for early diagnosis of diabetes. The IS group consisted of 52 healthy IS control subjects who fulfilled the following inclusion criteria:

• fasting plasma glucose <6.0 mmol/l(24), normal glucose tolerance, 120-min plasma glucose after 75-g oral glucose load <7.0 mmol/l(no diagnosis of type 1 or type 2 diabetes)

• no family history of type 2 diabetes or gestational diabetes

To correct for the influence of the body weight and body fat mass on FFA and leptin levels, BMI matched subgroups from the IR (n = 12, 6 males and 6 females) and the IS (n = 10, 5 males and 5 females) group were compared.

The study was approved by the ethics committee of the University of Leipzig. All subjects gave written informed consent before participating in the study.

The OGTT was performed according to the criteria of the American Diabetes Association (24). The patients documented a high-carbohydrate diet 3 days before the OGTT. The OGTT was taken after an overnight fast with 75 g standardized glucose solution (Glucodex Solution 75 g; Merieux, Montreal, Canada). Venous blood samples were taken at 0, 60, 90, and 120 min for measurements of plasma glucose, insulin, and C-peptide.

Insulin sensitivity was determined with the hyperinsulinemic-euglcyemic clamp method (25). Subjects with a whole-body glucose uptake <40 μmol · kg^-1· min^-1 were defined as insulin resistant, and probands with a whole-body glucose uptake >50 μmol · kg^-1 ·min^-1 were defined as insulin sensitive according to previously described criteria (20). After an overnight fast and supine resting for 30 min, intravenous catheters were inserted into antecubital veins in both arms. One was used for the infusion of insulin and glucose; the other was used for the frequent sampling. After a priming dose of 1.2 nmol/m² insulin, the infusion with insulin(Actrapid 100 U/ml; Novo Nordisk, Bagsvaerd, Denmark) was started with a constant infusion rate of 0.28 nmol · m^-2 body surface· min^-1 and continued for 120 min. After 3 min, the variable 20% glucose infusion rate was added. The glucose infusion rate was adjusted during the clamp to maintain the blood glucose at 5.0 mmol/l. Bedside blood glucose measurements were performed every 5 min using the glucose dehydrogenase technique with Hemocue B (Hemocue, Angelholm, Sweden). Additional blood samples were taken after 60 min and during the steady state for the determination of glucose, insulin, C-peptide, angiotensin II,TNF-α, FFA, GH, IGF-I, IGF binding protein 3 (IGFBP-3), and leptin.

BMI was calculated as weight (kilograms) divided by height (meters)squared. Waist and hip circumferences were measured. Blood samples were taken after an overnight fast and after 30 min in the supine position to determine serum lipids, angiotensin II, TNF-α, FFA, GH, IGF-I, IGFBP-3, leptin,and standard laboratory parameters. Because GH and IGF-I secretion is highly variable, at least three measurements at different time points were performed. Plasma glucose was measured by the glucose oxidase method (ESAT 6660-2;Prüfgerätewerk Medingen, Dresden, Germany). Plasma insulin was determined in a two-site chemiluminescent enzyme immunometric assay for the Immulite automated analyzer(Diagnostic Products, Los Angeles, CA). Plasma C-peptide was determined in a solid-phase chemiluminescent enzyme immunoassay using the Immulite automated analyzer. For the quantification of serum FFAs, an in vitro enzymatic colorimetric method was used (NEFA C, ACS-ACOD method; Wako, Neuss, Germany). Leptin was determined by a competitive in-house RIA for leptin. Polyclonal antibodies against human recombinant leptin were raised in rabbits (Peprotech,Rocky Hill, NJ). Standards or serum specimens in duplicate were mixed with 0.05 ml ¹²⁵I-labeled leptin and incubated with leptin antibody(diluted 1:10,000) for 16-20 h at 4°C. A mixture of anti-rabbit IgG and PEG 6000 was added for the double-antibody precipitation method. The sensitivity of the RIA (2 SD of the 0 ng/ml level, n = 12) was 0.2 ng/ml. Interassay and intra-assay coefficients of variation were <12.5% in the range between 1 and 8 ng/ml leptin. The recovery of dilution experiments(undiluted until 1:20) was 88-112% for the concentration range between 4 and 6 ng/ml. Leptin levels of our in-house RIA (x) are comparable with data of a commercially available leptin RIA (y) from Mediagnost(Tübingen, Germany) in sera of normal-weight and adipose subjects: y = -0.13 + 0.96x (n = 92, r = 0.94, P < 0.0001). Because leptin levels are largely determined by BMI, the leptin levels measured in this study were adjusted to BMI using the SD score (SDS), as previously described(26). In addition, BMI-matched IR subgroups (BMI 26.2 ± 0.3 kg/m²) and IS subgroups (BMI 25.9 ± 0.2 kg/m²) were investigated.

Plasma levels of human GH were determined in a solid-phase two-site chemiluminescent enzyme immunometric assay for use with the Immulite automated analyzer (Diagnostic Products). Serum levels of IGF-I were measured after acid ethanol extraction by a competitive solid-phase immunoassay according to the method of Kratzsch et al.(27). That assay was modified by the use of biotin for labeling of IGF-I and streptavidineuropium (Wallac,Turku, Finland) for the detection of labeled molecules by time-resolved fluorescence. The sensitivity of the assay (2 SD of the 0 ng/ml level, n = 12) was <0.9 ng/ml. Intra-assay and interassay coefficients of variation were <10% in the range between 100 and 500 ng/ml. Serum levels of IGFBP-3 were determined by a commercially available enzyme-linked immunosorbent assay (ELISA) (DSL, Sinsheim, Germany). The sensitivity of that assay was found to be <0.8 ng/ml. Intra- and interassay coefficients were<12% in the range between 4 and 40 ng/ml. Angiotensin II plasma levels were determined by an RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). Plasma TNF-α concentrations were determined in triplicate using a commercial ELISA (human TNF-α OptEIA; PharMingen, San Diego, CA). The sensitivity of that assay is <1 pg/ml

Data are shown as mean ± SD. For the statistical analysis, plasma concentrations in the steady state of the euglycemic clamp were used. The calculation of insulin sensitivity in the steady state during the second hour of the euglycemic clamp was performed as described(25). Insulin sensitivity was determined as the glucose infusion rate during the steady state of the clamp divided by the steady-state insulin concentration, as previously described(28). All calculations and statistics were performed with SPSS for Windows (SPSS, Chicago). The differences between the groups were tested by one-way analysis of variance. In case the P value was <0.05, the groups were compared by the appropriate test (Student's t test for unpaired samples or theχ² test). A P value of <0.05 was regarded as significant. Correlations between variables were tested with Spearman's correlation test. A correlation coefficient (r) of P <0.05 was accepted as significant.

The clinical and biochemical characteristics of the probands are summarized in Table 1. The groups were matched for sex distribution and fasting plasma glucose. The fasting plasma glucose in the IS group was 4.81 ± 0.1 mmol/l and in the IR group 5.12 ± 0.2 mmol/l (P = 0.13)(Table 1). The IR group was significantly older than the IS group. The BMI was significantly higher in the IR group compared with the IS group (Table 1). There were no significant differences in BMI, fasting plasma glucose, and other glycemic parameters between the different sexes. To correct for the influence of body weight and body fat mass on plasma concentrations of the investigated parameters, BMI-matched IR and IS subgroups were compared. These groups were also matched for the waist-to-hip ratio as an estimate of the adipose tissue distribution. The clinical and biochemical characteristics of these subgroups are shown in Table 2. There were no significant differences between the IR and the IS groups for the serum levels of creatinine, urea, uric acid, sodium, potassium,chloride, bilirubin, LDL and HDL cholesterol, triglycerides, protein, and hematologic parameters. Patients with acute infections were excluded from the study. All subjects in the IR group had IGT, as defined by a 120-min OGTT glucose concentration between 7.8 and 11.1 mmol/l. All subjects in the IS group had normal glucose tolerance (120-min plasma glucose <7.8 mmol/l). There were no differences for the fasting or 30-min plasma glucose between the IR and the IS group. The 60-, 90-, and 120-min glucose levels were significantly higher in the IR compared with the IS group. The plasma insulin concentrations in response to the OGTT were higher at all time points in the IR group compared with the IS group. In the hyperinsulinemic-euglycemic clamp,the whole-body glucose uptake was significantly higher in the IS group (67.3± 5.2 μmol · kg^-1 · min^-1) than in the IR group (23.8 ± 4.3 μmol · kg^-1 ·min^-1) (Table 1). In parallel, we calculated insulin sensitivity according to the homeostasis model assessment (HOMA) for each proband. The results of the HOMA analysis correlate with the results obtained by euglycemic clamp (r = 0.91, P< 0.05). The BMI and the waist circumference correlate with the extent of insulin resistance.

There was no difference in the mean plasma TNF-α concentration between the IS group and the IR group (Table 1). There was no relation between the TNF-α level and age,sex, or BMI in any group. The mean plasma GH concentrations were not significantly different between the IS and the IR groups(Table 1). Basal IGF-I plasma concentrations were not different between the IR and the IS groups(Table 1). This lack of discrepancy also applies to the concentrations of the IGFBP-3(Table 1). There was no correlation between the age and the sex of the participants and the GH (mean from at least three measurements) and IGF-I levels. A correlation between the BMI and GH or IGF-I was not found. Furthermore, no statistically significant differences were found between the angiotensin II plasma concentrations in the IS and the IR groups (Table 1). There were no correlations between angiotensin II plasma concentrations and BMI or blood pressure.

The extent of insulin resistance determined by the whole-body glucose uptake did not correlate with the plasma concentration of TNF-α,angiotensin II, GH, or IGF-I (Table 3).

There is a strong positive relationship among the amount of adipose tissue,sex, and leptin levels (29). Therefore, serum leptin concentrations were adjusted for sex and BMI by calculating the SDS (26) and by comparing BMI-matched IR and IS subgroups. The calculated leptin SDS for females was 0.087 ± 0.03 in the IR group and -0.071 ± 0.04 in the IS group (NS); for males, it was 0.96 ± 0.18 in the IR group and 0.78 ± 0.08 in the IS group (NS). There were no significant differences for the serum leptin concentrations between the BMI-matched IR and IS subgroups (Fig. 1A). No correlation between the leptin levels in the BMI-matched groups and the extent of insulin resistance in the euglycemic clamp could be found(Table 3). There was a correlation between the fasting insulin levels and leptin levels (r =0.81). However, a significant correlation between leptin and insulin serum concentrations was not detectable in the BMI-matched subgroups.

Although the fasting FFA serum levels are in the normal range in both groups, the IR subjects had significantly (P < 0.05) higher serum concentrations of FFAs (0.59 ± 0.12 mmol/l) compared with the IS group(0.31 ± 0.08 mmol/l), independent of sex. To correct for the known association between BMI and FFA serum concentrations, BMI-matched IR and IS subgroups were compared. Independent of BMI, the IR subgroup had significantly higher FFA concentrations compared with the IS subgroup(Fig. 1B). The FFA serum concentrations correlate (r = 0.76) with the extent of insulin resistance (μmol · kg^-1 · min^-1), as determined by euglycemic clamps.

A number of physiological conditions (e.g.,puberty, pregnancy, and advanced age), genetic defects, or endocrine disorders are associated with insulin resistance(30). The determination of the primary metabolic defects leading to insulin resistance is important to understand the pathogenesis of type 2 diabetes. Because glucose toxicity of hyperglycemia per se impairs both insulin action and insulin secretion(31), the primary metabolic defects leading to insulin resistance should be investigated at an early stage of type 2 diabetes development. The early stages of type 2 diabetes development are characterized by a deterioration of glucose tolerance over years(22,24,32). IGT is an intermediate metabolic state between normal and diabetic glucose metabolism (24), and individuals with IGT are at high risk to develop type 2 diabetes. Therefore,obese individuals with IGT and insulin resistance reflected by a low whole-body glucose uptake during euglycemic clamp(Table 1) are a suitable subgroup for the investigation of the primary metabolic defects of type 2 diabetes.

We investigated the relationship between the potential inhibitors of IRS signaling (TNF-α, IGF-I, GH, and angiotensin II) and insulin resistance in individuals with IGT and severe insulin resistance at an early stage of diabetes development.

TNF-α has been shown to induce insulin resistance in vitro(33,34,35)and in animal models (36). In humans, the role of TNF-α in insulin resistance is still controversial. In one study, the administration of TNF-α led to insulin resistance(6), and TNF-αoverexpression in adipose tissue(37) and muscle(38) of obese IR subjects correlates with insulin resistance. However, Ofei et al.(39) did not find any effect of recombinant TNF-α-neutralizing antibody on insulin resistance in obese subjects with type 2 diabetes. In our study, there was no correlation between the plasma concentration of TNF-α determined by a highly sensitive assay and the degree of insulin resistance measured as whole-body glucose uptake during euglycemic clamp. One explanation for these results could be that local TNF-α production in adipose tissue is not released into the circulation and, therefore, does not alter TNF-α levels in peripheral blood. Thus, our results do not exclude that TNF-α acts in a paracrine or autocrine manner. The latter could in part explain the lack of insulin resistance improvement after administration of anti—TNF-αantibody (39). Moreover, an elevation of TNF-α plasma levels could occur at a later state of diabetes development secondary to further metabolic alterations(40), which are not present in our subjects.

Because angiotensin II negatively modulates insulin signaling by stimulating multiple serine phosphorylation events in the early components of the insulin signaling cascade in vitro(7), elevated angiotensin II serum concentrations are a possible cause for the coincidence of insulin resistance and hypertension. Folli et al.(7) showed that angiotensin II was able to inhibit the insulin-stimulated PI3K activity in the rat heart(mediated via the angiotensin₁ receptor) by 60%, suggesting that angiotensin II can be regarded as a potential inducer of insulin resistance. In healthy individuals, angiotensin II infusion increases insulin sensitivity determined by the glucose uptake during euglycemic clamp(41,42),but not in patients with type 2 diabetes(41). The different in vivo results can partly be explained by the existence of different angiotensin receptors or a higher affinity of the IRS cascade to insulin stimulation, as compared with the angiotensin II stimulation if both agents are simultaneously acting. In our study, there was no evidence that angiotensin II either increases or decreases insulin sensitivity, because individuals with extreme insulin resistance and healthy IS subjects had no significantly different angiotensin II plasma levels, and because no correlation between the glucose uptake in the euglycemic clamp and the angiotensin II plasma concentrations was detectable (Table 3). Because no participant had hypertension from our data, we cannot exclude that, in subjects with insulin resistance and hypertension, angiotensin II could secondarily cause a deterioration of insulin resistance. Therefore, elevated angiotensin II levels do not seem to be a primary metabolic defect in insulin resistance.

Elevated growth hormone plasma concentrations are associated with insulin resistance in patients with acromegaly(30) or in GH-deficient patients treated with GH (10)or during GH therapy in children with short stature(43). Because GH phosphorylates IRS proteins(4), GH could be a potential inhibitor of the IRS-signaling cascade. In our subjects, there is no evidence that elevation in GH plasma concentrations could be a primary defect in the insulin resistance syndrome. There were no differences in the mean GH levels between the IR and the IS group. The IS group has slightly increased but not significantly higher IGF-I plasma concentrations compared with the IR group. IGF-I was shown to improve insulin sensitivity in GH-deficient individuals(10), and tissue availability of circulating IGF-I seems to be a determinant of insulin sensitivity in patients with hypertension(44). We cannot exclude a decreased peripheral IGF-I tissue availability in the IR group. However, it is unlikely that alterations of the IGF-I serum concentration are a primary metabolic defect leading to insulin resistance.

In summary, elevated plasma concentrations of potential antagonists of the IRS cascade (TNF-α, IGF-I, GH, and angiotensin II) seem not to be the primary metabolic alteration of the early stages of the insulin resistance syndrome. This is also suggested by the observation that plasma levels of these parameters do not correlate with the BMI. However, these results do not allow concluding a cause-and-effect relationship between the assayed plasma concentrations of the parameters and insulin resistance on the cellular level in adipose tissue or skeletal muscle.

There were no significant differences in the leptin serum concentrations between the IR and the IS group after BMI matching(Fig. 1A) and after adjustment of the leptin concentrations for the BMI by the SDS(26). Therefore, increased serum leptin levels are unlikely to be a primary metabolic alteration in the development of type 2 diabetes. These results are in contrast to the recently reported elevation of leptin levels in offspring of patients with type 2 diabetes(18,19). A possible explanation for the different findings is that the leptin levels in our individuals were only normalized for the BMI, whereas in the other studies, leptin levels were normalized for total fat mass, intra-abdominal fat mass, and fasting plasma insulin levels.

Elevated FFA concentrations in different physiological situations(11,12,13)or in patients with type 2 diabetes(14) are associated with peripheral insulin resistance. Santomauro et al.(15) recently showed that overnight lowering of elevated FFAs with Acipimox improves insulin resistance in obese diabetic and nondiabetic subjects. Mason et al.(16) showed that prolonged elevation of plasma FFAs can desensitize the insulin secretory response to glucose in vivo, thereby inducing a β-cell defect similar to type 2 diabetes. FFA concentrations are commonly elevated in obesity(45). Therefore, we also compared BMI-matched IR and IS subgroups. Plasma FFA concentrations in the IR group were significantly higher than those in the IS group. Moreover, the plasma FFA levels correlated with the extent of insulin resistance(Table 1). Genetic defects are the most likely causes for increased FFA levels, potentially leading to insulin resistance in the IR group.

In conclusion, the plasma concentrations of TNF-α, GH, and angiotensin II are not elevated in patients with IGT and insulin resistance at an early stage of diabetes development, suggesting that alterations of these potential inhibitors of the IRS system are not the primary metabolic defects leading to insulin resistance. There was also no correlation between IGF-I and leptin serum concentrations and the extent of insulin resistance as measured by the glucose disposal in the euglycemic clamp. Elevated FFA levels could be an early metabolic defect in the insulin resistance syndrome.

This study was supported by a grant of the German Diabetes Association and the Interdisciplinary Center for Clinical Research at the University of Leipzig (IZKF B 15).

We thank H.-U. Häring, K. Rett, and E. Maerker for support to establish the hyperinsulinemic-euglycemic clamp technique for this study. We also thank Prof. Richter and R. Unger, Institute of Clinical Chemistry and Pathobio-chemistry, University of Leipzig, for the assistance in performing FFA assays. We would also like to thank all patients and volunteers for their participation in this study.