HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blüher, M. Articles by Paschke, R. Search for Related Content PubMed PubMed Citation Articles by Blüher, M. Articles by Paschke, R. Social Bookmarking                What's this?

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

From the III Medical Department (M.B., R.P.), Faculty of Medicine, and the Institute of Clinical Chemistry and Pathobiochemistry (J.K.), University of Leipzig, Leipzig, Germany.

Address correspondence and reprint requests to Prof. R. Paschke, University of Leipzig, III. Medical Department, Philipp-Rosenthal-Straße 27, D-04103 Leipzig, Germany. E-mail: pasr{at}medizin3.uni-leipzig.de .

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS CONCLUSIONS References

 
OBJECTIVE— To investigate the relationship between insulin resistance and plasma concentrations of free fatty acids (FFAs), leptin, and potential agonists of the insulin receptor substrate (IRS) system, including tumor necrosis factor- (TNF-), IGF-I, growth hormone (GH), and angiotensin II in individuals with impaired glucose tolerance (IGT).

RESEARCH DESIGN AND METHODS— Because glucose toxicity per se leads to insulin resistance, the determination of the primary metabolic alterations leading to insulin resistance is best accomplished in individuals who are at an increased risk to develop type 2 diabetes. Therefore, 48 subjects with IGT and insulin resistance (IR), characterized by hyperinsulinemic-euglycemic clamps, were compared with 52 healthy insulin-sensitive (IS) control subjects with respect to the relationship between the plasma levels of TNF-, IGF-I, GH, angiotensin II, FFA, leptin, and insulin resistance.

RESULTS— Between the IR and the IS groups, there were no significant differences in the plasma concentrations of TNF-, GH, angiotensin II, IGF-I, and leptin. However, plasma FFA levels were significantly elevated in the IR group compared with the IS group after matching for BMI.

CONCLUSIONS— The plasma concentrations of FFA, but not TNF-, IGF-I, GH, and angiotensin II, are elevated in patients at an early stage of insulin resistance, suggesting that FFAs, but not the other modulators of the IRS system, may be a primary metabolic abnormality leading to insulin resistance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS CONCLUSIONS References

 
Insulin resistance appears to play a pivotal role in the pathogenesis of type 2 diabetes and other chronic diseases, including hypertension, cardiovascular disease, and hyperlipidemia (1,2). In addition to the insulin receptor, there are a number of other receptors, including seven transmembrane receptors and cytokine/growth hormone (GH) receptors, that use the insulin receptor substrate (IRS) system for further intracellular signal transduction (3). Tumor necrosis factor- (TNF-), GH, angiotensin II, and IGF-I interact with these receptors and, therefore, could potentially inhibit or at least modulate the insulin-signaling pathway (4). Liu et al. (5) showed that TNF- inhibits insulin signaling in human adipocytes in vitro. Moreover, the administration of TNF- leads to insulin resistance (6). Angiotensin II stimulates tyrosine phosphorylation of IRS-1 and IRS-2, which leads to a binding of the IRSs to phosphatidylinositol 3-kinase (PI3K). Folli et al. (7) showed that the PI3K activity is inhibited by angiotensin II in a dose-dependent manner, suggesting that angiotensin II negatively modulates insulin signaling. Angiotensin II could therefore contribute to insulin resistance in individuals with increased activity of the renin-angiotensin system (7). Although IGF-I is considered to be the mediator of the growth-promoting effects of GH, the metabolic effects of these two hormones are different. Euglycemic clamp studies show that insulin sensitivity improves in patients with acromegaly after transsphenoidal adenomectomy (8), and in transgenic rabbits, overexpression of GH led to symptoms of insulin resistance (9). GH treatment in GH-deficient humans induces insulin resistance, whereas IGF-I administration to these subjects improves insulin sensitivity (10). Both GH and IGF-I engage the IRS system and could thereby affect the insulin-signaling pathway.

Free fatty acid (FFAs) and leptin are other mediators linked to insulin resistance that have unknown interactions with the insulin-signaling cascade. Elevation in FFA levels produces peripheral insulin resistance in a concentration-dependent manner in healthy nonpregnant (11,12), pregnant (13), and diabetic subjects (14); likewise, overnight lowering of elevated FFAs with acipimox improves insulin resistance in obese diabetic and nondiabetic subjects (15). Prolonged elevation of FFAs induces a ß-cell defect similar to that found in type 2 diabetes (16). Moreover, in Zucker diabetic fatty (ZDF) rats, FFAs have been shown to induce apoptosis of ß-cells via de novo ceramide formation and increased nitric oxide production (17). High serum leptin levels are associated with the insulin-resistant (IR) phenotype in offspring of patients with type 2 diabetes (18,19). However, the underlying mechanisms for the elevated leptin levels in IR subjects remain to be determined.

A number of studies have investigated the defects in glucose metabolism in healthy first-degree offspring of patients with type 2 diabetes (20,21). By means of a euglycemic clamp and an intravenous glucose tolerance test with respect to the early alterations in glucose metabolism, Weyer et al. (22,23) characterized Pima Indians with impaired glucose tolerance (IGT) or impaired fasting glucose with a high risk to develop diabetes. However, in these studies, alterations in plasma levels of TNF-, GH, angiotensin II, IGF-I, FFA, and leptin have not been investigated.

In summary, TNF-, GH, angiotensin II, IGF-I, FFA, and leptin can induce insulin resistance. There is no study investigating these parameters in relation to insulin resistance determined by the euglycemic clamp in prediabetic IR individuals. Because hyperglycemia per se can induce insulin resistance, only subjects with normal fasting blood glucose levels were chosen for the study. Our aims were to investigate whether there are differences in the plasma concentration of these parameters between a well-characterized pheno-type with severe insulin resistance and a group of healthy insulin-sensitive (IS) control subjects without a family history of type 2 diabetes and to determine if any of these parameters could serve as a marker for the severity of insulin resistance.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS CONCLUSIONS References

 
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.

OGTT
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.

Hyperinsulinemic-euglycemic glucose clamp
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-, FFA, GH, IGF-I, IGF binding protein 3 (IGFBP-3), and leptin.

Assays and calculations
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-, 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- concentrations were determined in triplicate using a commercial ELISA (human TNF- OptEIA; PharMingen, San Diego, CA). The sensitivity of that assay is <1 pg/ml

Statistical analysis
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 the ² 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.

	RESULTS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS CONCLUSIONS References

 
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.

Plasma concentrations of TNF-, IGF-I, GH, and angiotensin II
There was no difference in the mean plasma TNF- concentration between the IS group and the IR group (Table 1). There was no relation between the TNF- 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-, angiotensin II, GH, or IGF-I (Table 3).

Leptin and FFAs
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.

View larger version (27K): [in this window] [in a new window]   Figure 1 —A: Plasma leptin concentrations in BMI-matched IR and IS subgroups. Regardless of the extent of insulin resistance, female subjects had generally higher leptin plasma concentrations than male subjects. There were no significant differences in leptin levels between the IR and the IS subgroup. B: Plasma FFA concentrations in BMI-matched IR and IS subgroups. The probands in the IR group had significantly higher FFA concentrations compared with the IS group. *P < 0.05.

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.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS CONCLUSIONS References

 
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-, 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- has been shown to induce insulin resistance in vitro (33,34,35) and in animal models (36). In humans, the role of TNF- in insulin resistance is still controversial. In one study, the administration of TNF- led to insulin resistance (6), and TNF- overexpression 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--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- 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- production in adipose tissue is not released into the circulation and, therefore, does not alter TNF- levels in peripheral blood. Thus, our results do not exclude that TNF- acts in a paracrine or autocrine manner. The latter could in part explain the lack of insulin resistance improvement after administration of anti—TNF- antibody (39). Moreover, an elevation of TNF- 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-, 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 ß-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-, 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.

	ACKNOWLEDGMENTS

 
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.

	FOOTNOTES

 
M.B. is currently affiliated with the Research Division, Joslin Diabetes Center, Boston, Massachusetts.

Abbreviations: ELISA, enzyme-linked immunosorbent assay; FFA, free fatty acid; GH, growth hormone; HOMA, homeostasis model assessment; IGFBP-3, IGF binding protein 3; IGT, impaired glucose tolerance; IR, insulin-resistant; IRS, insulin receptor substrate; IS, insulin-sensitive; OGTT, oral glucose tolerance test; PI3K, phosphatidylinositol 3-kinase; RIA, radioimmunoassay; SDS, SD score; TNF-, tumor necrosis factor-.

A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

Received for publication August 9, 2000. Accepted for publication November 1, 2000.

	References

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS CONCLUSIONS References

 

1. Reaven GM: Role of insulin resistance in human disease. Diabetes 37:1595 -1607, 1988[Abstract]
   
2. DeFronzo RA, Ferranini E: Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care34 : 416-422,1991
   
3. Myers MG Jr, White MF: Insulin signal transduction and the IRS proteins. Annu Rev Pharmacol Toxicol36: 615-658,1996[Medline]
   
4. Yenush L, White MF: The IRS-signalling system during insulin and cytokine action. BioEssays 19:491 -500, 1997[Medline]
   
5. Liu LS, Spelleken M, Rohring K, Hauner H, Eckel J: Tumor necrosis factor alpha acutely inhibits insulin signalling in human adipocytes: implication of the p80 tumor necrosis factor receptor. Diabetes 47:515 -522, 1998[Abstract]
   
6. van der Pol T, Romijn JA, Endert E, Borm JJ, Büller HR, Sauerwein HP: TNF mimics the metabolic response of acute infections in healthy humans. Am J Physiol 261:E457 -E465, 1991[Abstract/Free Full Text]
   
7. Folli F, Saad MJA, Velloso L, Hansen H, Caradente O, Feener EP, Kahn CR: Crosstalk between insulin and angiotensin II signalling systems. Exp Clin Endocrinol Diabetes107 : 133-139,1999[Medline]
   
8. Wasada T, Aoki K, Sato A, Katsumori K, Muto K, Tomonaga O, Yokoyama H, Iwasaki N, Babazono T, Takahashi C, Iwamoto Y, Omori Y, Hizuka N: Assessment of insulin resistance in acromegaly associated with diabetes mellitus before and after transsphenoidal adenomectomy. Endocr J 44: 617-620,1997[Medline]
   
9. Costa C, Solanes G, Visa J, Bosch F: Transgenic rabbits overexpressing growth hormone develop acromegaly and diabetes mellitus. FASEB J 12:1455 -1460, 1998[Abstract/Free Full Text]
   
10. Hussain MA, Schmitz O, Mengel A, Glatz Y, Christiansen JS, Zapf J, Froesch ER: Comparison of the effects of growth hormone and insulin-like growth factor I on substrate oxidation and on insulin sensitivity in growth hormone-deficient humans. J Clin Invest94 : 1126-1233,1994
    
11. Boden G, Chen X, Ruiz J, White JV, Rossetti L: Mechanisms of fatty acid-induced inhibition of glucose uptake. J Clin Invest 93:2438 -2446, 1994
    
12. Roden M, Price TB, Perseghin G, Petersen KF, Rothman DL, Cline GW, Shulman GI: Mechanisms of fatty acid-induced insulin resistance in humans. J Clin Invest 97:2859 -2865, 1996[Medline]
    
13. Sivan E, Homko CJ, Whittaker PG, Reece EA, Boden G: Free fatty acid and insulin resistance during pregnancy. J Clin Endocrinol Metab 83:2338 -2342, 1998[Abstract/Free Full Text]
    
14. Boden G, Chen X: Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes. J Clin Invest 96:1261 -1268, 1995
    
15. Santomauro AT, Boden G, Silva ME, Rocha DM, Santos RF, Ursich MJ, Strassmann PG, Wajchenberg BL: Overnight lowering of free fatty acids with Acipimox improves insulin resistance and glucose tolerance in obese diabetic and nondiabetic subjects. Diabetes48 : 1836-1841,1999[Abstract]
    
16. Mason TM, Goh T, Tchipashvili V, Sandhu H, Gupta N, Lewis GF, Giacca A: Prolonged elevation of plasma free fatty acids desensitizes the insulin response to glucose in vivo in rats. Diabetes48 : 524-530,1999[Abstract]
    
17. Shimabukuro M, Zhou YT, Levi M, Unger RH: Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci U S A 95:2498 -2502, 1998[Abstract/Free Full Text]
    
18. Vauhkonen I, Niskanen L, Haffner S, Kainulainen S, Uusitupa M, Laakso M: Insulin resistant phenotype is associated with high serum leptin levels in offspring of patients with non-insulin-dependent diabetes mellitus. Eur J Endocrinol 139:598 -604, 1998[Abstract]
    
19. Nyholm B, Fisker S, Lund S, Moller N, Schmitz O: Increased circulating leptin concentrations in insulin-resistant first-degree relatives of patients with non-insulin-dependent diabetes mellitus: relationship to body composition and insulin sensitivity but not to family history of non-insulin-dependent diabetes mellitus. Eur J Endocrinol 136:173 -179, 1997[Abstract]
    
20. Vauhkonen I, Niskanen L, Vanninen E, Kainulainen S, Uusitupa M, Laakso M: Defects in insulin secretion and insulin action in non-insulin-dependent diabetes mellitus are inherited. J Clin Invest 100:86 -96, 1997
    
21. Volk A, Renn D, Overkamp B, Mehnert E, Maerker E, Jacob S, Balletshofer B, Häring HU, Rett K: Insulin action and secretion in healthy, glucose tolerant first degree relatives of patients with type 2 diabetes mellitus: influence of body weight. Exp Clin Endocrinol Diabetes107 : 140-147,1999[Medline]
    
22. Weyer C, Bogardus C, Pratley RE: Metabolic characteristics of individuals with impaired fasting glucose and/or impaired glucose tolerance. Diabetes 48:2197 -2203, 1999[Abstract]
    
23. Weyer C, Bogardus C, Mott DM, Pratley RE: The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest104 : 787-794,1999[Medline]
    
24. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus: Report the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care21 (Suppl. 1): S5-S19,1998
    
25. DeFronzo RA, Tobin JD, Andres R: Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 237:E214 -E223, 1979[Abstract/Free Full Text]
    
26. Blum WF, Englaro P, Hanitsch S, Juul A, Hertel NT, Müller J, Skakkebaek NE, Heiman ML, Birkett M, Attanasio AM, Kiess W, Rascher W: Plasma leptin levels in healthy children and adolescents: dependence on body mass index, body fat mass, pubertal stage, and testosterone. J Clin Endocrinol Metab82 : 2904-2910,1997[Abstract/Free Full Text]
    
27. Kratzsch J, Blum WF, Schenker E, Keller E, Jahreis G, Haustein B, Ventz M, Rotzsch W: Measurements of insulin-like growth factor I (IGF-I) in normal adults, patients with liver cirrhosis and acromegaly: experience with new competitive enzyme immunoassay. Exp Clin Endocrinol 101:144 -149, 1993[Medline]
    
28. Larsson H, Ahren B: Insulin resistant subjects lack islet adaptation to short-term dexamethason-induced reduction in insulin sensitivity. Diabetologia 42:936 -943, 1999[Medline]
    
29. Caro JF, Sinha MK, Kolaczynski JW, Zhang PL, Considine RV: Leptin: the tale of an obesity gene. Diabetes45 : 1455-1462,1996[Medline]
    
30. Tritos NA, Mantzoros CS: Syndromes of severe insulin resistance. J Clin Endocrinol Metab 83:3025 -3030, 1998[Free Full Text]
    
31. Rossetti L, Giaccari A, DeFronzo RA: Glucose toxicity. Diabetes Care 13:610 -630, 1990[Abstract]
    
32. DeFronzo RA: Lilly Lecture 1987. The triumvirate: ß-cell, muscle, liver: a collusion responsible for NIDDM. Diabetes 37:667 -687, 1988[Medline]
    
33. Stephens JM, Lee J, Pilch PF: Tumor necrosis factor alpha induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT-4 expression without a loss of insulin-receptor-mediated transduction. J Biol Chem272 : 971-976,1997[Abstract/Free Full Text]
    
34. Hotamisligil GS, Budavari A, Murray D, Spiegelman BM: Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes: central role of tumor necrosis factor alpha. J Clin Invest94 : 1543-1549,1994
    
35. Hotamisligil GS, Murray D, Choy LN, Spiegelman BM: Tumor necrosis factor alpha inhibits signalling from the insulin receptor. Proc Natl Acad Sci U S A 91:4854 -4858, 1994[Abstract/Free Full Text]
    
36. Cheung AT, Ree D, Kolls JK, Fuselier J, Coy DH, Bryer-Ash M: An in vivo model for elucidation of the mechanism of tumor necrosis factor- (TNF)-induced insulin resistance: evidence for differential regulation of insulin signalling by TNF. Endocrinology139 : 4928-4935,1998[Abstract/Free Full Text]
    
37. Hotamisligil GS, Arner P, Caro JF, Atkinson L, Spiegelman BM: Increased adipose tissue expression of tumor necrosis factor alpha in human obesity and insulin resistance. J Clin Invest95 : 2409-2415,1995
    
38. Saghizadeh M, Ong JM, Garvey WT, Henry RR, Kern PA: The expression of TNF alpha by human muscle: relationship to insulin resistance. J Clin Invest 97:1111 -1116, 1996[Medline]
    
39. Ofei F, Hurel S, Newkirk J, Sopwith M, Taylor R: Effects of engineered human anti—TNF- antibody (CDP571) on insulin sensitivity and glycemic control in patients with NIDDM. Diabetes 45:881 -885, 1996[Abstract]
    
40. Zinman B, Hanley AJG, Harris SB, Kwan J, Fantus IG: Circulating tumor necrosis factor- concentrations in a native Canadian population with high rates of type 2 diabetes. J Clin Endocrinol Metab 84:272 -278, 1999[Abstract/Free Full Text]
    
41. Fliser D, Keller C, Bahrmann P, Franek E, Schreckling H, Bergis K, Ritz E: Altered action of angiotensin II in patients with type 2 diabetes mellitus of recent onset. J Hypertens15 : 293-299,1997[Medline]
    
42. Widgren BR, Urbanavicius V, Wikstrand J, Attvall S, Persson B: Low-dose angiotensin II increases glucose disposal rate during euglycemic hyperinsulinemia. Am J Hypertens6 : 892-895,1993[Medline]
    
43. Heptulla RA, Boulware SD, Caprio S, Silver D, Sherwin RS, Tamborlane WV: Decreased insulin sensitivity and compensatory hyperinsulinemia after hormone treatment in children with short stature. J Clin Endocrinol Metab 82:3234 -3238, 1997[Abstract/Free Full Text]
    
44. Laviades C, Gil MJ, Monreal I, Gonzalez A, Diez J: Tissue availability of insulin-like growth factor I is inversely related to insulin resistance in essential hypertension: effects of angiotensin-converting enzyme inhibition. J Hypertens 16:863 -870, 1998[Medline]
    
45. Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD: Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020 -1024, 1988[Abstract]
    

CiteULike

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Bluher, J. W. Bullen Jr., J. H. Lee, S. Kralisch, M. Fasshauer, N. Kloting, J. Niebauer, M. R. Schon, C. J. Williams, and C. S. Mantzoros Circulating Adiponectin and Expression of Adiponectin Receptors in Human Skeletal Muscle: Associations with Metabolic Parameters and Insulin Resistance and Regulation by Physical Training J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2310 - 2316. [Abstract] [Full Text] [PDF]

				R. M. Krauss Lipids and Lipoproteins in Patients With Type 2 Diabetes Diabetes Care, June 1, 2004; 27(6): 1496 - 1504. [Abstract] [Full Text] [PDF]

				T. D. Wiesner, M. Bluher, M. Windgassen, and R. Paschke Improvement of Insulin Sensitivity after Adrenalectomy in Patients with Pheochromocytoma J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3632 - 3636. [Abstract] [Full Text] [PDF]

				J. Krakoff, T. Funahashi, C. D.A. Stehouwer, C. G. Schalkwijk, S. Tanaka, Y. Matsuzawa, S. Kobes, P. A. Tataranni, R. L. Hanson, W. C. Knowler, et al. Inflammatory Markers, Adiponectin, and Risk of Type 2 Diabetes in the Pima Indian Diabetes Care, June 1, 2003; 26(6): 1745 - 1751. [Abstract] [Full Text] [PDF]

				P. A. Lee, S. D. Chernausek, A. C. S. Hokken-Koelega, and P. Czernichow International Small for Gestational Age Advisory Board Consensus Development Conference Statement: Management of Short Children Born Small for Gestational Age, April 24-October 1, 2001 Pediatrics, June 1, 2003; 111(6): 1253 - 1261. [Abstract] [Full Text] [PDF]

				S. Dzienis-Straczkowska, M. Straczkowski, M. Szelachowska, A. Stepien, I. Kowalska, and I. Kinalska Soluble Tumor Necrosis Factor-{alpha} Receptors in Young Obese Subjects With Normal and Impaired Glucose Tolerance Diabetes Care, March 1, 2003; 26(3): 875 - 880. [Abstract] [Full Text] [PDF]

				M. Straczkowski, I. Kowalska, A. Stepien, S. Dzienis-Straczkowska, M. Szelachowska, and I. Kinalska Increased Plasma-Soluble Tumor Necrosis Factor-{alpha} Receptor 2 Level in Lean Nondiabetic Offspring of Type 2 Diabetic Subjects Diabetes Care, October 1, 2002; 25(10): 1824 - 1828. [Abstract] [Full Text] [PDF]

				S. Franckhauser, S. Munoz, A. Pujol, A. Casellas, E. Riu, P. Otaegui, B. Su, and F. Bosch Increased Fatty Acid Re-esterification by PEPCK Overexpression in Adipose Tissue Leads to Obesity Without Insulin Resistance Diabetes, March 1, 2002; 51(3): 624 - 630. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blüher, M. Articles by Paschke, R. Search for Related Content PubMed PubMed Citation Articles by Blüher, M. Articles by Paschke, R. Social Bookmarking                What's this?