Regulation of adiponectin production by insulin: interactions with tumor necrosis factor-α and interleukin-6

Abstract

Obesity is often associated with insulin resistance, low-grade systemic inflammation, and reduced plasma adiponectin. Inflammation is also increased in adipose tissue, but it is not clear whether the reductions of adiponectin levels are related to dysregulation of insulin activity and/or increased proinflammatory mediators. In this study, we investigated the interactions of insulin, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in the regulation of adiponectin production using in vivo and in vitro approaches. Plasma adiponectin and parameters of insulin resistance and inflammation were assessed in a cohort of lean and obese insulin-resistant subjects. In addition, the effect of insulin was examined in vivo using the hyperinsulinemic-euglycemic clamp, and in adipose tissue (AT) cultures. Compared with lean subjects, the levels of total adiponectin, and especially the high-molecular-weight (HMW) isomer, were abnormally low in obese insulin-resistant subjects. The hyperinsulinemic clamp data confirmed the insulin-resistant state in the obese patients and showed that insulin infusion significantly increased the plasma adiponectin in lean but not obese subjects (P < 0.01). Similarly, insulin increased total adiponectin release from AT explants of lean and not obese subjects. Moreover, expression and secretion of TNF-α and IL-6 increased significantly in AT of obese subjects and were negatively associated with expression and secretion of adiponectin. In 3T3-L1 and human adipocyte cultures, insulin strongly enhanced adiponectin expression (2-fold) and secretion (3-fold). TNF-α, and not IL-6, strongly opposed the stimulatory effects of insulin. Intriguingly, the inhibitory effect of TNF-α was especially directed toward the HMW isomer of adiponectin. In conclusion, these studies show that insulin upregulates adiponectin expression and release, and that TNF-α opposes the stimulatory effects of insulin. A combination of insulin resistance and increased TNF-α production could explain the decline of adiponectin levels and alterations of isomer composition in plasma of obese insulin-resistant subjects.

Figures

Fig. 1.

Total and adiponectin isomers in plasma of lean and morbidly obese subjects. Analysis of plasma adiponectin isomers was performed with ELISA, using an adiponectin multimeric assay kit from Alpco Immunoassays, as described in materials and methods. Results are presented as means ± SE. Statistical differences between lean and obese subjects were performed by unpaired Student's t-test. Significant differences are indicated by asterisks: **P < 0.01, *P < 0.05.

Fig. 2.

Relationship of plasma insulin levels in lean and obese subjects. A: effect of varying insulin concentrations on plasma adiponectin levels in lean (L, n = 7) and obese (O, n = 21) subjects undergoing a 2-step (each for 2 h) hyperinsulinemic-euglycemic clamp; basal levels were obtained after a 12-h fast prior to insulin infusion. Steady-state low (3- to 4-fold; step 1) and high (8- to 10-fold basal; step 2) insulin levels were achieved following insulin infusions of 0.6–1.0 and 2–3.0 mU·kg−1·min−1, each for 2 h while maintaining euglycemia using a variable infusion of 20% dextrose. Values are means ± SE. Statistical differences between obese and lean for each time point are presented with an asterisk: *P < 0.001. Differences between values before (basal) and after insulin infusion for each group are indicated by letter (aP < 0.01). Correlations are shown between plasma insulin and adiponectin levels in lean (B) and obese (C) subjects during the euglycemic-hyperinsulinemic clamps. Pearson correlation coefficient is shown for lean subjects; there was no significant correlation for obese subjects.

Fig. 3.

Adiponectin secretion from subcutaneous (Sc) and omental (Om) adipose tissue cultures. Sc adipose tissues of lean (A) and obese (B) subjects were pretreated (24 h) with insulin (INS) or vehicle (CNT), and adiponectin secretion was tested and compared with control nontreated explants (CNT). Adiponectin secretions (24 h) in Sc and Om fats are shown for lean (A) and obese subjects (B). Analysis of adiponectin isomers in culture medium (D) was performed by Western blotting followed by separation in a nondenaturing polyacrylamide gel electrophoresis condition as described in materials and methods. Representative blot and mean of optic density are shown in C and D, respectively. Data are presented as means ± SE; n = 6 for lean subjects (BMI 24 ± 1 kg/m2) and n = 12 for obese subjects (BMI 45 ± 5 kg/m2). Statistical differences between insulin-treated (INS) and control (CNT) for the same fat depot and the same subject group are indicated by asterisks: ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig. 4.

Secretion rates of IL-6 (A and B) and TNF-α (C and D) from Sc and Om adipose tissue cultures. Adipose tissue explants of lean (A and C) and obese (B and D) subjects were pretreated (24 h) with insulin or vehicle, and adiponectin secretion was tested for the subsequent 24 h as described in materials and methods. Data are presented as means ± SE; n = 6 for lean subjects and n = 12 for obese subjects. Statistical differences between fat depots of lean and obese subjects for the same treatment (CNT vs. CNT and INS vs. INS) are indicated by letter: aP < 0.001.

Fig. 5.

Expression of adiponectin (A and F), TNF-α (B and G), IL-6 (C and H), AdipoR1 (D and I) and AdipoR2 (E and J) in Sc and Om adipose tissue of lean (L) and obese (O) subjects: Data for qPCR are calculated as relative expression of genes and represent ΔCT (cycle threshold) normalized to β-actin as described in materials and methods. Data are presented as means ± SE; n = 13 for lean subjects (BMI: 23.8 ± 1.2 kg/m2) and n = 36 for obese subjects (BMI: 45 ± 9 kg/m2). Statistical analysis was performed by Student's t-test. Differences between lean and obese subjects for the same fat depots are indicated by asterisks: ***P < 0.001 and **P < 0.01. Differences between Sc and Om of obese subjects are indicated by letters: aP < 0.01 and bP < 0.05. Relations between expression of adiponectin and TNF-α (log) and IL-6 (log) are presented in K and L, respectively. Pearson correlation coefficients are shown.

Fig. 6.

Adiponectin secretion and expression in human adipocytes. Conditions for isolation and differentiation of human adipocytes are detailed in materials and methods. Adipocytes were pretreated for 24 h with 100 nM insulin or vehicle (A). Aliquots of medium were harvested at indicated times for the measurement of adiponectin secretion. Adiponectin expression was examined at the end of the experiment by qPCR (B). Statistical analysis was performed with Student's t-test. Differences between insulin-treated (INS) and nontreated cells (CNT) are indicated with asterisks: **P < 0.001 and *P < 0.05. Differences between INS + TNF-α treatment and INS treatment are indicated by letter: aP < 0.01.

Fig. 7.

Total adiponectin secretion and expression in 3T3-L1 adipocytes. Culture and treatments of adipocytes were performed according to the conditions described in materials and methods. Results are presented as secretion rates per 8 h. A: adiponectin secretion after incubation with insulin or vehicle. B: effects of IL-6 alone or with insulin (INS + IL-6). C: effects of TNF-α alone or with insulin (INS + TNF-α). D: effects of these treatments on adiponectin expression measured by qPCR as indicated in materials and methods. Results are presented as means ± SE. Statistical differences between CNT and INS (A and D) are indicated by asterisk: **P < 0.001. Statistical differences between combined treatments (INS + IL-6 or TNF-α) and insulin treatment (INS) (B and C vs. A) are indicated with lower-case letters: aP < 0.001 and bP < 0.01. Statistical difference between combined treatment (INS + IL-6 or TNF-α) and single treatment with either IL-6 or TNF-α are indicated with upper-case letters: AP < 0.01 and BP < 0.05.

Fig. 8.

Effect of insulin and cytokines on adiponectin isomer composition in 3T3-L1 adipocytes. Conditions of culture and treatments were performed as described in materials and methods. Proteins of culture medium were separated by nondenaturing electrophoresis, and Western blotting was performed using specific anti-adiponectin antibody as described in materials and methods. Representative blot and means of optic density are shown in A and B, respectively. Data are presented as means ± SE; n = 3 experiments, each performed in triplicate. Statistical difference between combined treatment (INS + IL-6 or INS + TNF-α) and insulin treatment (INS) are indicated by asterisk: **P < 0.01 and *P < 0.05.