Adipose tissue promotes a serum cytokine profile related to lower insulin sensitivity after chronic central leptin infusion

Obesity is an inflammatory state characterized by an augment in circulating inflammatory factors. Leptin may modulate the synthesis of these factors by white adipose tissue decreasing insulin sensitivity. We have examined the effect of chronic central administration of leptin on circulating levels of cytokines and the possible relationship with cytokine expression and protein content as well as with leptin and insulin signaling in subcutaneous and visceral adipose tissues. In addition, we analyzed the possible correlation between circulating levels of cytokines and peripheral insulin resistance. We studied 18 male Wistar rats divided into controls (C), those treated icv for 14 days with a daily dose of 12 μg of leptin (L) and a pair-fed group (PF) that received the same food amount consumed by the leptin group. Serum leptin and insulin were measured by ELISA, mRNA levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, IL-10 and tumor necrosis factor-α (TNF-α) by real time PCR and serum and adipose tissue levels of these cytokines by multiplexed bead immunoassay. Serum leptin, IL-2, IL-4, IFN-γ and HOMA-IR were increased in L and TNF-α was decreased in PF and L. Serum leptin and IL-2 levels correlate positively with HOMA-IR index and negatively with serum glucose levels during an ip insulin tolerance test. In L, an increase in mRNA levels of IL-2 was found in both adipose depots and IFN-γ only in visceral tissue. Activation of leptin signaling was increased and insulin signaling decreased in subcutaneous fat of L. In conclusion, leptin mediates the production of inflammatory cytokines by adipose tissue independent of its effects on food intake, decreasing insulin sensitivityThis work was supported by grants from Fondo de Investigación Sanitaria (PI10/0747), Ministerio de Ciencia y Tecnología (SAF 2010-22277), CIBERobn (CB03/06) and Fundación Endocrinología y Nutrición. S.C. is supported by CIBERob

Insulin signaling in subcutaneous and visceral fat. A.

<p>Relative levels of the insulin receptor beta chain (IRβ) in inguinal fat of rats that received saline (C) or chronic leptin (L) and the pair-fed group (PF). <b>B.</b> Relative IRβ levels in epididymal fat of the same groups. <b>C.</b> Relative phosphorylated (p) Akt on serine 473 (pSer473-Akt) protein levels in inguinal fat of the same groups. <b>D.</b> Relative pSer473-Akt protein levels in epididymal fat of the same groups. <b>E.</b> Relative levels of the forkhead box-containing protein O-1 (FOXO1) in inguinal fat of the same groups. <b>F.</b> Relative FOXO1 levels in epididymal fat of the same groups. <b>G.</b> Relative levels of the protein tyrosine phosphatase 1B (PTP1B) in inguinal fat of the same groups. <b>H.</b> Relative PTP1B levels in epididymal fat of the same groups. The data are expressed as a percentage of the control ratio. DU, densitometry units; NS, non-significant; *<i>p</i><0.05 by ANOVA, ***<i>p</i><0.001 by ANOVA.</p

Homeostasis model assessment of insulin resistance (HOMA-IR), insulin sensitivity as delta (Δ) in glycemia after intracerebroventricular insulin infusion (ICVII) and glycemia during an intraperitoneal insulin tolerance test (IPITT). A.

<p>HOMA-IR index in rats that received saline (C) or chronic leptin (L) and pair-fed group (PF). <b>B.</b> Delta (Δ) in glycemia ([glycemia after 120 min of insulin bolus] – [glycemia before insulin bolus]) in rats that received saline plus acute insulin (insulin, C+I), chronic leptin plus acute insulin (L+I) and the pair-fed group plus insulin (PF+I). <b>C.</b> Serum glucose levels before (0 min) and during (30, 60, 90 and 120 min) an IPITT. *<i>p</i><0.05, **<i>p</i><0.01 by ANOVA.</p

General characteristics of experimental animals. A.

<p>Mean daily food intake in rats that received saline (C) or chronic leptin (L) and the pair-fed group (PF). <b>B.</b> Mean body weight measurements throughout the study in the same groups. <b>C.</b> Epididymal fat content in the same groups. <b>D.</b> Serum leptin concentrations in the same groups. <b>E.</b> Serum glucose concentrations in the same groups. <b>F.</b> Serum insulin concentrations in the same groups. NS, non-significant; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.01 by ANOVA. * in panels A and B indicates significant difference (<i>p</i><0.05) between C and L or PF groups, whereas ^# indicates significant difference (<i>p</i><0.05) between PF and L groups.</p

Relative mRNA and protein content of cytokines in subcutaneous adipose tissue. A.

<p>Relative mRNA levels of interleukin-2 (IL-2) in inguinal fat of rats that received saline (C) or chronic leptin (L) and pair-fed group (PF). <b>B.</b> Protein content of IL-2 in the same groups. <b>C.</b> Relative mRNA levels of interferon-γ (IFN-γ) in the same groups. <b>D.</b> Protein content of IFN-γ in the same groups. <b>E.</b> Relative mRNA levels of tumor necrosis factor α (TNF-α) in the same groups. <b>F.</b> Protein content of TNF-α in the same groups. NS, non-significant; *<i>p</i><0.05 by ANOVA.</p

Linear correlations of homeostasis model assessment of insulin resistance (HOMA-IR), Δ glycemia ([glycemia after 120 min of insulin bolus] – [glycemia before insulin bolus]) after intracerebroventricular insulin infusion (ICVII) and area under the curve (AUC) after intraperitoneal insulin tolerance test (IPITT) with serum levels of leptin, interleukin (IL)-2 and IL-4.

<p>r, correlation coefficient; NS, non-significant; *<i>p</i><0.05, **<i>p</i><0.01.</p

Serum levels of cytokines. A.

<p>Serum interleukin (IL)-2 levels in rats that received saline (C) or chronic leptin (L) and the pair-fed group (PF). <b>B.</b> Serum IL-4 levels in the same groups. <b>C.</b> Serum IL-6 levels in the same groups. <b>D.</b> Serum IL-10 levels in the same groups. <b>E.</b> Serum interferon γ (IFN-γ) levels in the same groups. <b>F.</b> Serum tumor necrosis α (TNF-α) levels in the same groups. NS, non-significant; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 by ANOVA.</p

Serum cytokine levels (pg/ml), insulin resistance or sensitivity indexes, relative mRNA levels and concentration of cytokines in subcutaneous and visceral adipose tissue (pg/mg of protein) and intracellular signaling (expressed as % control) in both fat depots.

<p>FOXO1, forkhead box O number 1; HOMA-IR, homeostasis model assessment of insulin resistance; IL, interleukin; IFN-γ, interferon-γ; IR, insulin receptor; ObRb, long form of leptin receptor; p/t, phosphorylated/total; PTP1B, protein tyrosine phosphatase 1B; SOCS3, suppressor of cytokine signaling 3; STAT3, transducer and activator of transcription factor 3; TNF-α, tumor necrosis factor α. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 by ANOVA vs. control; ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001 by ANOVA vs. pair-fed group.</p

Relative mRNA and protein content of cytokines in visceral adipose tissue. A.

<p>Relative mRNA levels of interleukin-2 (IL-2) in epididymal fat of rats that received saline (C) or chronic leptin (L) and the pair-fed group (PF). <b>B.</b> Protein content of IL-2 in the same groups. <b>C.</b> Relative mRNA levels of interferon-γ (IFN-γ) in the same groups. <b>D.</b> Protein content of IFN-γ in the same groups. <b>E.</b> Relative mRNA levels of tumor necrosis factor α (TNF-α) in the same groups. <b>F.</b> Protein content of TNF-α in the same groups. NS, non-significant; *<i>p</i><0.05, ***<i>p</i><0.001 by ANOVA.</p