Advancing age and insulin resistance: role of plasma tumor necrosis factor-alpha

Giuseppe Paolisso1, Maria Rosaria Rizzo1, Gherardo Mazziotti2, Maria Rosaria Tagliamonte1, Antonio Gambardella1, Mario Rotondi2, Carlo Carella2, Dario Giugliano1, Michele Varricchio1, and Felice D'Onofrio1

1 Department of Geriatric Medicine and Metabolic Diseases and 2 Institute of Endocrinology-II, University of Napoli, 80138 Naples, Italy

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In 70 healthy subjects with a large age range, the relationships between plasma tumor necrosis factor-alpha (TNF-alpha ) and body composition, insulin action, and substrate oxidation were investigated. In the cross-sectional study (n = 70), advancing age correlated with plasma TNF-alpha concentration (r = 0.64, P < 0.001) and whole body glucose disposal (WBGD; r= -0.38, P < 0.01). The correlation between plasma TNF-alpha and age was independent of sex and body fat (BF; r = 0.31, P < 0.01). Independent of age and sex, a significant relationship between plasma TNF-alpha and leptin concentration (r = 0.29, P < 0.02) was also found. After control for age, sex, BF, and waist-to-hip ratio (WHR), plasma TNF-alpha was still correlated with WBGD (r = -0.33, P < 0.007). Further correction for plasma free fatty acid (FFA) concentration made the latter correlation no more significant. In a multivariate analysis, a model made by age, sex, BF, fat- free mass, WHR, and plasma TNF-alpha concentrations explained 69% of WBGD variability with age (P < 0.009), BF (P < 0.006), fat-free mass (P < 0.005), and plasma TNF-alpha (P < 0.05) significantly and independently associated with WBGD. In the longitudinal study, made with subjects at the highest tertiles of plasma TNF-alpha concentration (n = 50), plasma TNF-alpha concentration predicted a decline in WBGD independent of age, sex, BF, WHR [relative risk (RR) = 2.0; 95% confidence intervals (CI) = 1.2-2.4]. After further adjustment for plasma fasting FFA concentration, the predictive role of fasting plasma TNF-alpha concentration on WBGD (RR = 1.2; CI = 0.8-1.5) was no more significant. In conclusion, our study demonstrates that plasma TNF-alpha concentration is significantly associated with advancing age and that it predicts the impairment in insulin action with advancing age.

insulin action; substrate oxidation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE RELATIONSHIP between advancing age and impaired insulin action is well known (11, 29). Recently, tumor necrosis factor-alpha (TNF-alpha ) has been implicated in the development of insulin resistance (8, 19, 20, 34) and, therefore, in the pathogenesis of obesity (27, 32) and non-insulin-dependent diabetes mellitus (NIDDM) (1, 17). With regard to age, studies in mice have already shown plasma TNF-alpha concentration to increase with advancing age (3, 15, 26); nevertheless, data in humans are lacking. Age-related increase in body fatness might be responsible for an increase in plasma TNF-alpha concentration, which in turn might contribute to derange insulin action in the elderly.

Recently, leptin, the product of the ob/ob gene (35), has been considered a further potential candidate as a fat tissue-dependent mediator causing insulin resistance (22). If a cross talk between adipose tissue and skeletal muscle is mediated by TNF-alpha or leptin, an endocrine mechanism involving circulating TNF-alpha and leptin is conceivable. In light of such a possibility, a correlation among plasma TNF-alpha and leptin concentrations and insulin sensitivity should be found. Unfortunately, data on such associations are contrasting (14, 21). Furthermore, no study in a large sample of aged subjects has been made.

In light of such experimental evidence, we asked the following questions. 1) Does an age-related increase in plasma TNF-alpha concentration occur in humans? If the answer to this question is yes, 2) does plasma TNF-alpha concentration affect age-related insulin resistance? Finally, 3) is there any relationship between fasting plasma TNF-alpha and leptin concentrations in humans? To answer these questions, plasma TNF-alpha and leptin concentrations were measured and insulin-mediated glucose uptake and substrate oxidation were determined by euglycemic hyperinsulinemic glucose clamp and indirect calorimetry, respectively, in 70 healthy subjects with a wide age range.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Subjects and study design. Seventy subjects (44 males and 26 females) with a wide age range (21-94 yr) were studied. Premenopausal women were all studied in the luteal phase. All subjects were normotensive, took no medications, were nonsmokers, and had no evidence of metabolic or cardiovascular diseases. Furthermore, erythrocyte sedimentation rate, plasma fibrinogen and lactate deydrogenase isoenzymes, hemoglobin concentrations, and blood white cells were within normal range in all subjects. Oral glucose tolerance (75 g glucose) (34a) was tested in all volunteers before they were enrolled, and those affected by diabetes mellitus and glucose intolerance were excluded from the study. Subjects with a family history of NIDDM, obesity, or hypertension were also excluded from the study. All tests were conducted in the morning and after an overnight fast (of >= 12 h).

Study design consists of two parts, a cross-sectional study (n = 70), in which associations among all variables in the whole group of subjects were studied, and a longitudinal study (n = 50) with a 12-mo duration. In the cross-sectional study, subjects were categorized in relation to tertile of plasma TNF-alpha . Those subjects at the two highest tertiles of plasma TNF-alpha were enrolled for the longitudinal study. The reasons for such a choice were 1) to perform a longitudinal study only in subjects in whom an association between plasma TNF-alpha and whole body glucose disposal (WBGD) was already found in the cross-sectional study and 2) to have a reasonable number of subjects participating in the longitudinal study. At the end of the follow-up period, anthropometric characteristics, glucose tolerance, and insulin action were redetermined. Subjects had been on similar standard weight-maintaining diets containing 150 g of carbohydrate per day for >= 7 days before all tests.

After a clear explanation of the potential risks of the study, which was approved by the Ethical Committee of the University of Napoli, all volunteers provided informed consent to participate. More detailed characteristics of the subjects are reported in Table 1.

                              
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Table 1.   Clinical characteristics of the subjects

Anthropometric determinations. Weight and height were measured using a standard technique. Body fat (BF) and fat- free mass (FFM) were measured using a four-terminal bioimpedance analyzer (BIA; RJL Spectrum Bioelectrical Impedance-BIA 101/SC Akern, RJL-System, Florence, Italy) (5). Prediction of FFM by BIA was done with equations validated for a wide age range in the elderly (5). Waist circumference was measured at the midpoint between the lower rib margin and the iliac crest (normally umbilical level), and hip circumference was measured at the level of trochanter. Both circumferences were measured to the nearest 0.5 cm with a plastic tape, and the ratio of waist to hip (WHR) was calculated.

Metabolic tests. At baseline, blood samples for fasting plasma glucose, insulin, leptin, free fatty acids (FFA), triglycerides, and TNF-alpha concentrations were drawn. Insulin action was measured using the euglycemic hyperinsulinemic glucose clamp technique. In this test, a priming dose of insulin (154 pmol/kg) was given before the clamp was started. Then, a fixed insulin infusion (Humulin R, at the rate of 7.1 pmol · kg-1 · min-1 for 120 min; Eli Lilly, Florence, Italy) and a variable amount of glucose (as a 20% solution) were delivered. Simultaneous indirect calorimetry was performed using an open-circuit ventilated hood system (Deltatrac Monitor, Datex, Helsinki, Finland). Respiratory quotient and substrate oxidation rate were calculated from the oxygen consumption, the carbon dioxide production, and the nitrogen urinary excretion rate according to Ferrannini (10).

Analytic methods. Plasma glucose was immediately measured by the glucose oxidase method (Beckman Autoanalyzer; Fullerton, CA). Fasting plasma FFA concentrations were measured in triplicate on each sample, according to the method of Dole and Meinertz (7). Commercial enzymatic methods were used in the determination of plasma triglyceride (Peridecrome; Boehringer Mannheim, Milan, Italy) concentration. Blood samples for insulin, TNF-alpha , and leptin measurements were stored at -80°C until the assay measurements, which were all made in triplicate in one assay. Blood samples for insulin and TNF-alpha measurements were collected in heparinized tubes. After centrifugation, plasma insulin (Sorin Biomedical, Milan, Italy), leptin (Linco Research, St. Louis, MO), and TNF-alpha (Medgenix Diagnostic SA, Fleurus, Belgium) concentrations were determined by radioimmunoassay.

Calculations and statistical analyses. WBGD was calculated during the final 60 min of the clamp, as previously reported (11). In preliminary clamps, an insulin infusion rate of 7.1 pmol · kg-1 · min-1 fully suppressed (but without negative numbers) hepatic glucose output at all ages. Nonoxidative glucose metabolism (NOGM) was calculated as WBGD, and oxidative glucose metabolism was calculated by indirect calorimetry (10).

For predicting the adequacy of sample size in the cross-sectional study, an nQuery test was used. To approximate normal distribution, plasma insulin, leptin, and triglyceride concentrations were log transformed and used as such in all calculations. Univariate analysis allowed us to distribute all patients in tertiles of plasma TNF-alpha concentration and WBGD. The differences among each tertile were calculated by ANOVA. When ANOVA indicated a P <0.05, Scheffé's test was also performed. Pearson product-moment correlations were calculated to test association among variables. Partial correlations tested the association between two variables independent of a covariate. Multivariate linear regression analyses tested the independent association of each variable with plasma TNF-alpha concentration and WBGD. In the longitudinal study, the relative risk (RR) estimated the hazard of having a further decline in insulin-mediated glucose uptake for a hypothetical subject at the 75th and 25th percentiles of the risk factor in that subgroup, after adjustment for different covariates. For each RR, 95% confidence intervals (CI) are presented. Statistical analyses were performed by the SOLO (BMDP, Cork, Ireland) software package. All values are presented as means ± SD.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cross-sectional study. In the whole group of subjects (n = 70), advancing age was significantly associated with an increase in plasma TNF-alpha concentration (r = 0.64, P < 0.001) and a decline in WBGD (r = -0.38, P < 0.001; Fig. 1). Plasma leptin concentration was significantly correlated with BF (r = 0.71, P < 0.001) and WHR (r = 0.58, P < 0.001). Such correlations persisted after adjustment for age and gender (P < 0.008 for both). Simple correlations between plasma TNF-alpha concentration and main anthropometric and clinical variables studied are reported in Table 2. Plasma TNF-alpha concentration was positively correlated with BF (Fig. 2) and WHR, whereas no correlation with fasting plasma leptin concentration was found. After adjustment for sex and BF, the relationship between plasma TNF-alpha and age was weakened but still significant (r = 0.31, P < 0.01), whereas adjustment for age and sex made the relationship between plasma TNF-alpha and leptin concentration significant (r = 0.29, P < 0.02; Fig. 3). In contrast, the association between plasma TNF-alpha and leptin concentrations was no more significant after adjustment for BF (r = 0.11, P < 0.18). Plasma TNF-alpha concentrations were also correlated with fasting plasma glucose, insulin, triglyceride, and FFA concentrations (Table 2). Such correlations persisted even after adjustment for age, sex, BF, and WHR. Finally, a significant correlation between fasting plasma FFA and WBGD (r = -0.46, P < 0.001) was also found. In a multivariate analysis with plasma TNF-alpha concentration as a dependent variable, a model made by age, sex, BF, and WHR explained 47% of plasma TNF-alpha variability. In this model, only age (P < 0.008) and BF (P < 0.002) were significant determinants of plasma TNF-alpha concentration.


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Fig. 1.   Simple correlations between age and tumor necrosis factor-alpha (TNF-alpha ; r = 0.64, P < 0.001) and whole body glucose disposal (WBGD; r = -0.38, P < 0.001).

                              
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Table 2.   Simple correlations between TNF-alpha and main anthropometric and clinical variables


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Fig. 2.   Simple correlation between fasting plasma TNF-alpha and body fat (r = 0.45, P < 0.001).


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Fig. 3.   Partial correlation between fasting plasma TNF-alpha and leptin concentrations (r = 0.29, P < 0.02). All values were adjusted for age and sex.

During insulin infusion, steady-state plasma glucose (range: 4.8-5.0 mmol/l) and insulin (range: 560-600 pmol/l) concentrations were kept with narrow range [coefficient of variation (CV) <4.0%] without statistically significant differences between the sexes (P = 0.68). In these metabolic conditions, fasting plasma TNF-alpha concentration correlated with WBGD (r = -0.51, P < 0.001), insulin-stimulated glucose oxidation (r = -0.49, P < 0.001), and NOGM (r = -0.42, P < 0.001; Fig. 4). After control for age, sex, BF, and WHR, plasma TNF-alpha was still correlated with WBGD (r = -0.33, P < 0.007), insulin-stimulated glucose oxidation (r = -0.28, P < 0.02), and NOGM (r = -0.31, P < 0.01). After control for plasma FFA concentration, those correlations were not significant. Univariate analysis allowed us to divide the subjects in tertiles of age- and sex-adjusted WBGD. Subjects at the lowest tertile of WBGD also had the most elevated plasma TNF-alpha concentration (91.2 ± 11.5 pg/ml) compared with those at the 2nd (53.4 ± 10.4 pg/ml, P < 0.01) and 3rd tertiles (24.2 ± 7.8 pg/ml, P < 0.003). Interestingly, the correlation between WBGD and plasma TNF-alpha was strong in subjects at the 1st tertile (n = 20; r = -0.62, P < 0.003), weak in the 2nd (n = 28; r = -0.42, P < 0.03), and not significant at the 3rd (n = 22; r = -0.31, P < 0.09) tertile of WBGD. By univariate analysis, we divided the subjects in tertiles of fasting plasma TNF-alpha concentration (1st tertile: <44.0 pg/ml; 2nd tertile: 44.0-84.0 pg/ml; 3rd tertile: >84.0 pg/ml).


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Fig. 4.   Simple correlations between plasma TNF-alpha and glucose oxidative metabolism (GOX; r = -0.49, P < 0.001), nonoxidative glucose metabolism (NOGM; r = -0.42, P < 0.001), and WBGD (r = -0.51, P < 0.001).

In the multivariate analysis, a model made by age, sex, BF, FFM, WHR, and plasma TNF-alpha concentrations explained 69% of WBGD variability with age (P < 0.009), BF (P < 0.006), FFM (P < 0.01), and TNF-alpha (P < 0.05) significantly and independently associated with WBGD. Notwithstanding, this latter association was lost after addition of FFA to the model.

Baseline plasma TNF-alpha samples were reassayed to test the reproducibility of the assay. No differences in mean values (35.2 ± 3.8 vs. 34.8 ± 3.7 pg/ml, P = NS) and in intra-assay CV (4.7 ± 0.3 vs. 4.9 ± 0.3%, P = NS) between the first and the control assay, respectively, were found.

Longitudinal study. Clinical characteristics of the subjects participating to the longitudinal study (n = 50) at baseline and at the end of the follow-up period are reported in Table 3. At baseline, subjects had a mean age of 72.6 ± 9.3 yr, were not obese, had a prevalent central BF distribution, and had a mean fasting TNF-alpha concentration of 83.4 ± 16.6 pg/ml. No subjects became diabetic or glucose intolerant. After 12 mo, no significant changes in BF content and distribution vs. baseline values were found. Plasma TNF-alpha concentration at baseline predicts a further decline in insulin-mediated glucose uptake (RR = 2.2, CI = 1.3-2.5), insulin-stimulated oxidative glucose metabolism (RR = 1.8, CI = 1.0-2.5), and NOGM (RR = 1.6, CI = 1.0-2.8). The predictive effect of plasma TNF-alpha concentration on further decline in insulin-mediated glucose uptake was found to be independent of age, sex, BF, and WHR (RR = 2.0, CI = 1.2-2.4). The predictive role of fasting plasma TNF-alpha concentration on insulin-mediated glucose uptake (RR = 1.2, CI = 0.8-1.5), insulin-stimulated oxidative metabolism (RR = 1.0, CI = 0.6-1.8), and NOGM (RR = 1.1, CI = 0.8-1.7) was lost after further adjustment for fasting plasma FFA concentration.

                              
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Table 3.   Clinical characteristics of subjects at baseline and at end of follow-up period

In a subset of 29 patients of the 50 participating in the longitudinal study, plasma TNF-alpha concentration was also assayed at the end of the follow-up period. In those subjects, plasma TNF-alpha concentration (75.5 ± 4.8 vs. 93.7 ± 5.2 pg/ml, P < 0.03) was more elevated after the follow-up period.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our study demonstrates that plasma TNF-alpha concentration is positively associated with advancing age and negatively correlated with insulin action and substrate oxidation. Furthermore, plasma TNF-alpha predicts a further worsening of insulin-mediated glucose uptake independent of age, sex, BF, and WHR but not of fasting plasma FFA concentrations. Interestingly, independent of age and sex, plasma TNF-alpha and leptin concentrations were also significantly correlated.

Why plasma TNF-alpha concentration increases with advancing age remains to be determined. Our volunteers were healthy subjects; thus a possible role of minor or major diseases, frequently occurring in the elderly, should be ruled out. Most likely, plasma TNF-alpha concentration parallels the age-related increase in body fatness. In our study, the relationship between plasma TNF-alpha and age was assessed only by a cross-sectional design, so a cause-effect relationship cannot be drawn. Nevertheless, the dependency of plasma TNF-alpha concentration by the age-related change in body composition can be drawn by inferred methods. In fact, the association between age and plasma TNF-alpha concentration was lost after adjustment for sex and BF; furthermore, BF was the major determinant of plasma TNF-alpha concentration, explaining 31% of its variability in the multivariate analysis with anthropometric characteristics of the subjects as independent variables.

Several factors have been suggested as contributing to age-related impairment of glucose disposal in skeletal muscle (11, 29). Elevated plasma TNF-alpha might provide a further contribution to the impairment of glucose metabolism in the elderly. In rats, TNF-alpha administration causes an increase in serum triglycerides and very low density lipoprotein (8). TNF-alpha -induced hyperlipidemia is thought to be the result of increased hepatic lipogenesis and lipolysis rather than of decreased peripheral clearance (8). In our study, the evidence that TNF-alpha may stimulate the lipolysis is strengthened by a significant correlation between plasma TNF-alpha and FFA concentrations. With regard to glucose metabolism, in vitro data showing the negative impact of plasma TNF-alpha on glucose metabolism are very consistent (2, 4, 13, 18, 30, 31, 33). Briefly, obese rats treated with antibodies against the TNF-alpha receptor (that is, IgG) were two to three times more sensitive to insulin than untreated rats (27), the effect being evident at skeletal muscle level and null at hepatic site. Furthermore, TNF-alpha has been shown to downregulate GLUT-4 mRNA levels in adipocytes and myocyte cultures (4, 27). In fat cells, this effect occurs in the context of downregulation of expression of several fat-specific genes, such as a P2 or adipsin (27, 33), so it is not entirely specific. Treatment of insulin-sensitive cells with TNF-alpha can clearly alter the catalytic activity of the insulin receptor. In adipocytes, TNF-alpha treatment leads to a reduction of insulin-stimulated receptor autophosphorylation and a more pronounced effect on insulin receptor substrate-1 phosporylation (18). Interestingly, Groder et al. (13) recently confirmed that TNF-alpha inhibits insulin receptor autophosphorylation and can act at receptor and postreceptor levels. Saghizadeh et al. (30) also demonstrated that, in muscular tissue of insulin-resistant patients, expression of TNF-alpha was fourfold higher than in subjects with normal insulin sensitivity; furthermore, an inverse linear relationship between maximal glucose disposal rate and degree of skeletal muscle expression of TNF-alpha was also observed. Finally, Miles et al. (25) recently demonstrated that TNF-alpha infusion in rats impairs insulin action, a phenomenon prevented by troglidazone administration.

Data in vivo are contrasting. The infusion of TNF-alpha might impair insulin action through a rise of counterregulatory hormone concentrations, such as glucagon, glucorticoids, and catecholamines (23). Kellerer et al. (21) demonstrated that insulin sensitivity was not a determinant of circulating TNF-alpha independent of age, gender, and percent desirable body weight in offspring of NIDDM patients. Ofei et al. (28) did not find an improvement in insulin action with use of a TNF-alpha neutralizing agent in humans. Frittitta et al. (12) demonstrated that WBGD negatively correlated with adipose PC-1 protein content but not with TNF-alpha gene expression. Our results are in agreement with the studies showing a negative impact of plasma TNF-alpha on insulin sensitivity, as demonstrated by the following observations. 1) There was a negative correlation between plasma TNF-alpha concentration and WBGD. 2) Plasma TNF-alpha concentration was an independent determinant of WBGD in multivariate analysis and explains 21% of WBGD variability. 3) Plasma TNF-alpha concentration negatively correlated with insulin-stimulated oxidation and NOGM, thus supporting the hypothesis that the cytokine deranges both receptor and postreceptor steps of glucose metabolism. 4) In the longitudinal study, plasma TNF-alpha predicts a further impairment in WBGD and substrate oxidation independent of age, sex, BF, and WHR. Why in vivo studies are not so consistent as in vitro studies is unknown. In our study the association between plasma TNF-alpha and WBGD was absent in subjects at the 3rd tertile of WBGD, whereas such a correlation was significant in subjects at the 1st and 2nd tertiles of WBGD. One can hypothesize that, in vivo, only elevated plasma TNF-alpha concentrations might have significant effect on WBGD. Alternatively, one could hypothesize that TNF-alpha might have a local effect on the muscle that is embedded with fat cells secreting this peptide. This does not necessarily exclude the importance of plasma levels as an indicator for greater local activity. Nevertheless, only studies performing a dose-effect curve might provide a response to such possibility. Finally, a negative impact of plasma TNF-alpha on insulin action might be due to an overactivity of the glucose-fatty acid cycle. In fact, in both cross-sectional and longitudinal studies, the relationship between TNF-alpha and WBGD was lost after adjustment for plasma FFA concentration.

In our study, we also observe a significant correlation between plasma TNF-alpha and leptin concentration that is independent of age and sex. Despite the fact that a correlation does not support a pathophysiological link, our data are in agreement with other results (9, 24) also showing a relationship between TNF-alpha and leptin. One can hypothesize that BF, having a coordinate control on plasma TNF-alpha and leptin concentration, might drive the relationship between these latter two variables. The facts that plasma leptin concentration differs with gender (16) and that plasma TNF-alpha concentration increases with advancing age might provide an explication for the need to adjust for age and sex to get a significant correlation.

In conclusion, our study demonstrates that plasma TNF-alpha concentration increases with advancing age and that such an increase is associated with an impairment in insulin-mediated glucose uptake and substrate oxidation with advancing age.

    FOOTNOTES

Address for reprint requests: G. Paolisso, Dept. of Geriatric Medicine and Metabolic Diseases, Servizio di Astanteria Medica, Piazza Miraglia 2, 80138 Napoli, Italy.

Received 31 December 1997; accepted in final form 24 April 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(2):E294-E299
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