Advertisement
Research Article

Adipose Tissue Promotes a Serum Cytokine Profile Related to Lower Insulin Sensitivity after Chronic Central Leptin Infusion

  • Emma Burgos-Ramos,

    Affiliations: Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa and Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain, Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain

    X
  • Sandra Canelles,

    Affiliations: Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa and Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain, Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain

    X
  • Arancha Perianes-Cachero,

    Affiliation: Grupo de Neurobioquímica, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Spain

    X
  • Eduardo Arilla-Ferreiro,

    Affiliation: Grupo de Neurobioquímica, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Spain

    X
  • Jesús Argente,

    Affiliations: Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa and Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain, Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain

    X
  • Vicente Barrios mail

    vbarrios@telefonica.net

    Affiliations: Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa and Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain, Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain

    X
  • Published: October 02, 2012
  • DOI: 10.1371/journal.pone.0046893

Abstract

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 sensitivity.

Introduction

Obesity is associated with an inflammatory state involved in the pathogenesis of many obesity related comorbidities. Previous findings indicate that inflammatory diseases mediate energy and weight deregulation though different proinflammatory cytokines [1], [2], whose levels are increased in both the circulation and peripheral tissues [3]. These changes predispose an individual to the development of type 2 diabetes mellitus, with this disease being associated with total and visceral obesity [4], [5].

Leptin modulates food intake, body weight and adipose stores, with a direct correlation between serum leptin levels, gene expression leptin in adipocytes and body fat [6]. Non-adipose cells are considered to be responsible for the production of the majority of proinflammatory factors [7], but adipocytes also synthetizes several cytokines [8]. Leptin also regulates immune function, playing a role in starvation-induced immunosuppression [9]. Deficient leptin signaling impairs cellular responses, whereas immune and malnutrition-related diseases are associated with increased synthesis of leptin and of inflammatory cytokines. In fact, leptin stimulates the production of proinflammatory cytokines by monocytes, largely distributed in the adipose tissue [10].

Hyperleptinemia is associated with insulin resistance. Although leptin initially increases insulin sensitivity, long-term exposure to high leptin levels has been reported to result in insulin resistance [11]. Leptin is a mediator of the inflammatory response that impairs insulin signaling in the hypothalamus and adipocytes [12], [13]. This inflammatory state favours the release of macrophage chemoattractant proteins, triggering insulin resistance that in turn induces a subsequent increase in circulating cytokines and fatty acids, leading to a lipotoxic state in non-adipose tissues that aggravates the pathological situation [14]. In addition, insulin resistance increases inflammatory cytokine synthesis in adipocytes, contributing to the exacerbation of this state [15].

The effect of exogenous leptin on insulin's actions and metabolic outputs has been studied mainly in leptin-deficient patients, as well as in models of experimental diabetes or obesity [11], [16]. However, there is little information in normal animals regarding the effect of leptin on the expression of proinflammatory cytokines in adipose tissue. The fact that leptin decreases food intake must also be kept in mind since the amount of food consumed may alter insulin sensitivity and the cytokine profile [17], [18], making it important to discriminate between the direct effects of leptin from those due to decreased food intake.

In the present study, we investigated how chronic exposure to increased leptin levels could modify the systemic cytokine profile and insulin resistance in a non-obese model. To discriminate between the direct effects of leptin and its induction of reduced food intake, a group of pair-fed rats was analyzed. The potential contribution of subcutaneous and visceral adipose tissues to the modifications in the cytokine profile was also examined.

Results

General characteristics of experimental groups

Food intake and body weight were recorded to verify that icv leptin infusion affected these parameters. On the fourth day of treatment, food intake was reduced in L and PF with respect to C (Fig. 1A); whereas the appearance of differences in body weight in L was found on the eighth day with respect to the C and PF groups (Fig. 1B). Epididymal fat mass was reduced in both PF and L, with this reduction being greater in L (Fig. 1C). Serum leptin levels were increased in L (Fig. 1D). Glycemia (Fig. 1E) and serum insulin levels (Fig. 1F) showed no significant differences among the experimental groups.

thumbnail

Figure 1. General characteristics of experimental animals. A.

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

doi:10.1371/journal.pone.0046893.g001

Chronic leptin administration changed serum proinflammatory cytokine levels, increased HOMA-IR index and attenuated the central and peripheral insulin effects on glycemia

Serum levels of IL-2, IL-4 and IFN-γ were increased in L with respect to C and PF (Fig. 2A, B and E, respectively). Interleukin-6 and -10 levels showed no differences among the groups (Fig. 2C and D, respectively), whereas TNF-α levels were reduced in PF and L (Fig. 2F).

thumbnail

Figure 2. Serum levels of cytokines. A.

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

doi:10.1371/journal.pone.0046893.g002

As inflammation is correlated with insulin resistance [19] we first calculated the homeostasis model assessment of insulin resistance (HOMA-IR). This insulin-related resistance index was increased in L compared to C and PF (Fig. 3A). To examine if HOMA-IR shows a relationship with serum cytokine values, linear correlation regressions were performed. There was a positive correlation of HOMA-IR with serum leptin, IL-2 and IL-4 levels (Table 1).

thumbnail

Figure 3. 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.

HOMA-IR index in rats that received saline (C) or chronic leptin (L) and pair-fed group (PF). 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). C. Serum glucose levels before (0 min) and during (30, 60, 90 and 120 min) an IPITT. *p<0.05, **p<0.01 by ANOVA.

doi:10.1371/journal.pone.0046893.g003
thumbnail

Table 1. 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.

doi:10.1371/journal.pone.0046893.t001

As icv insulin administration increases glycemia [12], [20], we also evaluated insulin sensitivity by measuring changes in serum glucose levels after central insulin infusion. Although blood glucose levels were in the normal physiological range in all groups throughout the study, previous chronic exposure to leptin reduced the rise in glycemia induced by icv insulin injection (Fig. 3B). Negative correlations of delta of glycemia with serum leptin, IL-2 and IL-4 levels were found (Table 1).

As central leptin may modify peripheral insulin response, we also investigated whether chronic icv leptin infusion would be accompanied by reduced insulin tolerance. Although we found no differences in basal glycemia, a rapid drop of glycemia was observed throughout the ip insulin tolerance test (IPITT) in control and pair-fed rats, whereas only a modest reduction of glucose levels was detected in leptin-treated rats (Fig. 3C). Linear regression analyses showed that HOMA-IR presented a direct correlation with serum cytokine levels, whereas the correlation between the Δ in glycemia and cytokine levels was negative. Finally, the relationship of the area under the curve for glucose (AUC) after the IPITT was negative with leptin and IL-2 whereas no significant correlation with was observed with IL-4 levels (Table 1).

Effect of leptin on relative mRNA and protein levels of cytokines in subcutaneous and visceral adipose tissues

To determine whether adipose tissue contributes to the generation of the systemic inflammatory profile found in leptin-treated rats, we studied relative messenger RNA levels of IL-2, IL-4, IFN-γ TNF-α in subcutaneous and visceral adipose tissues. IL-2 mRNA levels were increased in subcutaneous and visceral tissues of L (Fig. 4A and 5A, respectively), whereas mRNA levels of IFN-γ only increase in visceral adipose tissue (Fig. 5C). No changes in the mRNA levels of TNF-α were seen in either tissue (Fig. 4E and 5E). Messenger RNA levels of IL-4 were very low and could not be adequately quantified.

thumbnail

Figure 4. Relative mRNA and protein content of cytokines in subcutaneous adipose tissue. A.

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. Protein content of IL-2 in the same groups. C. Relative mRNA levels of interferon-γ (IFN-γ) in the same groups. D. Protein content of IFN-γ in the same groups. E. Relative mRNA levels of tumor necrosis factor α (TNF-α) in the same groups. F. Protein content of TNF-α in the same groups. NS, non-significant; *p<0.05 by ANOVA.

doi:10.1371/journal.pone.0046893.g004

Circulating cytokine levels are the result of the synthesis and liberation by several tissues. We analyzed protein concentrations of IL-2, IL-4, IFN-γ TNF-α in subcutaneous and visceral adipose tissue. IL-2 protein levels in subcutaneous adipose tissue were not different between the three groups (Fig. 4B), but was decreased in L with respect to C and PF in visceral adipose tissue (Fig. 5B). In L protein levels of IFN-γ were increased in both adipose depots (Fig. 4D and 5D) and TNF-α decreased in visceral adipose tissue (Fig. 5F), with no change in subcutaneous fat (Fig. 4F, Table 1). Finally, protein concentrations of IL-4 were increased in the subcutaneous adipose tissue of L with respect to C and PF groups (6.12±0.87, 5.76±1.09 and 8.94±0.61, expressed in pg of IL-4/mg of protein in C, PF and L groups, respectively), whereas no changes in visceral adipose tissue were seen (11.44±2.37, 10.44±1.06 and 10.54±0.92, expressed in pg of IL-4/mg of protein in C, PF and L groups, respectively).

thumbnail

Figure 5. Relative mRNA and protein content of cytokines in visceral adipose tissue. A.

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. Protein content of IL-2 in the same groups. C. Relative mRNA levels of interferon-γ (IFN-γ) in the same groups. D. Protein content of IFN-γ in the same groups. E. Relative mRNA levels of tumor necrosis factor α (TNF-α) in the same groups. F. Protein content of TNF-α in the same groups. NS, non-significant; *p<0.05, ***p<0.001 by ANOVA.

doi:10.1371/journal.pone.0046893.g005

Immune and inflammatory markers in the different adipose compartments

As specific cell infiltration could affect the reported inflammatory profile, we have analyzed several markers, expressed in different cell types. The levels of F4/80 are undetectable in subcutaneous and visceral adipose tissue of controls; however, a strong signal was detected in both fat depots of L group. Vimentin was increased in subcutaneous compartment of L (Fig. 6A) and decreased in visceral adipose tissue of L group (6B). Finally, levels of haptoglobin were increased in subcutaneous (Fig. 6C) and visceral adipose tissue (Fig. 6D), showing a direct correlation with IL-2 in both localizations (Fig. 6E and 6F).

thumbnail

Figure 6. Immune and inflammatory markers in adipose compartments. A.

Relative vimentin protein levels in inguinal fat of rats that received saline (C) or chronic leptin (L) and the pair-fed group (PF). B. Relative vimentin protein levels in epididymal fat of the same groups. C. Haptoglobin levels in inguinal fat of the same groups. D. Haptoglobin levels in epididymal fat of the same groups. E. Linear regression analysis between interleukin-2 (IL-2) and haptoglobin in inguinal fat. F. Linear regression analysis between interleukin-2 (IL-2) and haptoglobin in epididymal fat. Correlation coefficients (r) and p values are represented for each analysis. DU, densitometry units; *p<0.05 by ANOVA, ***p<0.001 by ANOVA.

doi:10.1371/journal.pone.0046893.g006

Intracellular signaling pathways of leptin and insulin in adipose tissue after leptin and insulin infusion

Leptin mRNA levels were increased in subcutaneous fat of L (Fig. 7A), with no change in visceral fat (Fig. 7B). The mRNA levels of ObRb were increased in subcutaneous adipose of L (Fig. 7C) and in visceral fat of PF (Fig. 7D). Phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Ser727 was increased in the subcutaneous adipose tissue of L (Fig. 7E), whereas in visceral tissue it was increased in both PF and L, with this increase being greater in PF (Fig. 7F). Levels of suppressor of cytokine signaling 3 (SOCS3) were unchanged in subcutaneous and visceral fat (Fig. 7G and 7H, respectively and Table 2).

thumbnail

Figure 7. Leptin signaling in subcutaneous and visceral fat. A.

Relative mRNA levels of leptin in inguinal fat of rats that received saline (C) or chronic leptin (L) and the pair-fed group (PF). B. Relative mRNA levels of leptin in epididymal fat of the same groups. C. Relative mRNA levels of the long form of the leptin receptor (ObRb) in inguinal fat of the same groups. D. Relative mRNA levels of ObRb in epididymal fat of the same groups. E. Relative phosphorylated (p) signal transducer and activator of transcription factor 3 phosphorylated on serine 727 (pSer727-STAT3) protein levels in inguinal fat of the same groups. F. Relative pSer727-STAT3 protein levels in epididymal fat of the same groups. G. Relative suppressor of cytokine signaling 3 (SOCS3) protein levels in inguinal fat of the same groups. H. Relative SOCS3 protein 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; *p<0.05 by ANOVA **p<0.01 by ANOVA.

doi:10.1371/journal.pone.0046893.g007
thumbnail

Table 2. 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.

doi:10.1371/journal.pone.0046893.t002

To determine if chronic exposure to increased leptin levels modulates insulin signaling, we examined insulin receptor β chain (IRβ) levels in subcutaneous and visceral tissues. Levels of IRβ in subcutaneous fat were not different between the experimental groups (Fig. 8A) and were reduced in visceral fat of L with respect to C and PF (Fig. 8B). Phosphorylation of Akt at Ser 473 was reduced in subcutaneous and visceral adipose of PF and L (Fig. 8C and 8D, respectively). No differences were detected in phosphorylation of this target between PF and L in subcutaneous tissue (Fig. 8C), whereas in visceral adipose tissue, the levels of Akt phosphorylation were lower in L compared to PF (Fig. 8D).

thumbnail

Figure 8. Insulin signaling in subcutaneous and visceral fat. A.

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. Relative IRβ levels in epididymal fat of the same groups. C. Relative phosphorylated (p) Akt on serine 473 (pSer473-Akt) protein levels in inguinal fat of the same groups. D. Relative pSer473-Akt protein levels in epididymal fat of the same groups. E. Relative levels of the forkhead box-containing protein O-1 (FOXO1) in inguinal fat of the same groups. F. Relative FOXO1 levels in epididymal fat of the same groups. G. Relative levels of the protein tyrosine phosphatase 1B (PTP1B) in inguinal fat of the same groups. H. 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; *p<0.05 by ANOVA, ***p<0.001 by ANOVA.

doi:10.1371/journal.pone.0046893.g008

We have determined Akt activation after insulin infusion in both fat pads. Phosphorylation of Akt at Ser 473 was reduced in subcutaneous adipose tissue of leptin-treated rats (100.0±7.9 vs. 44.8±4.1, p<0.01; in C plus insulin and L plus insulin, respectively), whereas no differences were detected in visceral fat (100.0±20.2 vs. 119.3±14.7, p = 0.79; in C plus insulin and L plus insulin, respectively) after central insulin infusion. We also analyzed Akt activation after IPIIT in both compartments. Phosphorylation of Akt at Ser 473 was decreased in subcutaneous adipose tissue (100.0±9.7 vs. 57.0±6.5, p<0.05; in C plus insulin and L plus insulin, respectively) as in visceral fat (100.0±11.2 vs. 75.6±3.4, p<0.05; in C plus insulin and L plus insulin, respectively).

Levels of forkhead box-containing protein O-1 (FOXO1) were increased in subcutaneous fat of L and visceral fat of PF and L (Fig. 8E and 8F, respectively, Table 2). Protein tyrosine phosphatase 1B (PTP1B) was increased in L respect to C and PF in subcutaneous adipose tissue (8G) and diminished in visceral depot of L group (8H).

Discussion

This study demonstrates that chronic central leptin administration causes a serum inflammatory profile correlated to insulin resistance. Adipose tissue contributes to the rise in circulating cytokine levels in leptin-treated rats and this increase does not seem to be correlated to the lower insulin sensitivity in adipose tissue. A direct effect of increased leptin levels exists as serum levels of most cytokines did not differ between pair-fed rats and controls.

Leptin induced a proinflammatory profile in serum, as previously shown [21], consistent with an elevation of inflammatory cytokines and leptin in obese patients [22]. There is increasing evidence that leptin enhances proinflammatory immune responses in different blood cell types, some of which are infiltrated in adipose tissue [23]. In addition, correlations between serum levels of leptin and several interleukins, as well as with IFN-γ, have been shown in several pathologies associated with inflammation [24], [25], [26]. In spite of an increase in several proinflammatory cytokines, serum TNF-α levels were reduced after leptin infusion. However, this decline is most likely not a direct effect of leptin, as similar TNF-α level were found in pair-fed rats. In fact, reduced food intake may have some anti-inflammatory effects and a low caloric diet not only ameliorates serum proinflammatory profiles, but also decreases the mRNA expression of up-regulated chemokines in obese patients [27].

Our experimental model reproduces a low-grade peripheral inflammatory situation without obesity, but where hyperleptinemia is present. We and others have reported that this experimental model of leptin infusion induces weight loss, central changes in leptin and insulin signaling and hyperleptinemia [12], [28]. Although we cannot discard the contribution of exogenous leptin to the rise in serum levels, leptin synthesis in subcutaneous adipose tissue is augmented and this may contribute to the increase in circulating levels. The mechanism by which an increase in central leptin levels stimulates leptin expression in fat depots is not well known, but it has been reported that central leptin infusion stimulates triiodothyronine production [29], [30]. This thyroid hormone increases the expression of leptin mRNA in subcutaneous fat depots [31] and also in adipocytes in vitro [32]. Although the main role of leptin is to regulation body weight by affecting food intake [33], it has additional effects on carbohydrate and lipid metabolism [34] that could explain the differences in weight gain between pair-fed and leptin-treated rats. Additionally, weight loss could also be potentiated by the levels of systemic cytokines [35].

Previous studies have demonstrated the relationship between obesity, hyperleptinemia and reduced insulin sensitivity and the beneficial effect of weight loss on insulin action [36], [37]. Our model produces a low-grade peripheral inflammation without obesity where hyperleptinemia is present and there is a correlation between leptin and several interleukins with peripheral insulin resistance. This interesting finding has been previously reported in patients where plasma leptin levels correlate with HOMA-IR independently of the effect of obesity [38], indicating a key role of hyperleptinemia per se in the generation of this adverse profile and suggesting that the contribution of adipose tissue may be more closely related to its functional state, than to its total amount. In addition, a direct effect of leptin has been shown in clinical studies where induced hyperleptinemia contributes to increase insulin resistance, whereas interruption of treatment ameliorates the sensitivity to insulin [11]. We have assessed insulin sensitivity after insulin challenge by using a central insulin bolus, because this infusion increased glycemia [12], [39]. The attenuated response in L suggest that chronic exposure to icv leptin decreased central insulin control of glucose levels [20], although we cannot exclude an effect of leptin treatment on peripheral tissues. Finally, we also evaluated insulin sensitivity after a classical intraperitoneal insulin tolerance test, observing a lower decrease in serum glucose levels in leptin-treated rats, thus suggesting reduced insulin sensitivity. Taken together, these results seem to indicate that leptin modify the response to insulin administration by decreasing its sensitivity.

Two of the main targets of leptin are STAT3 and SOCS3. In adipose tissue activation of STAT3 was coincident with increased expression of the leptin receptor. Although the direct action of leptin on the expression of its receptor is controversial, a positive effect has been previously reported [40], as we described here in inguinal adipose tissue. In contrast to subcutaneous adipose, in epididymal fat leptin receptor mRNA levels were increased in pair-fed rats. The positive effect of caloric restriction on leptin receptors has been reported [41], suggesting that moderate food reduction improves leptin signaling in some localizations. Hence, depot-specific differences have been reported both in experimental animals [42] and lean and obese patients [43].

Leptin modulates the production of interleukins in different cell types, including T cells and macrophages [44], [45] and generates an inflamed state in adipose tissue, probably regulated by crosstalk between adipocytes and macrophages [46], [47]. As mentioned above, the main findings reported here are the increase in mRNA levels of IL-2 in both inguinal and epididymal fat pads, the marked augment in IFN-γ mRNA in epididymal fat, and particularly, the increased pSTAT3 levels without changes in SOCS3 in subcutanteous fat in response to chronic leptin administration. The lack of leptin-induced STAT3 signaling is associated with reduced cytokine production [48], whereas leptin exposure increases both STAT3 activation and chemokine expression in macrophages [10]. Thus, the activation of leptin signaling reported here may account for the rise in the mRNA levels of cytokines. Not all changes in mRNA levels were related with changes in protein levels in adipose tissue, which is most likely due to increased secretion that would contribute to the generation of the serum inflammatory profile. However, the relationship is direct for some cytokines, such as IFNγ in visceral adipose. In contrast, TNF-α gene expression and levels in adipose tissue do not show an apparent relationship with serum profile. We must keep in mind that reduced food intake reduces white adipose tissue and leptin selectively decreases visceral adiposity [49], as we report here. Thus, although there are no changes in TNF-α mRNA levels per gram of adipose tissue, a decrease in the total amount of adipose tissue could contribute to the reduction of serum TNF-α.

Obesity and hyperleptinemia are associated with a proinflammatory state, with infiltration of different cell types, such as macrophages into adipose tissue [7]. The presence of the macrophage marker F4/80 after chronic leptin infusion indicates the presence of these cells in both fat depots that could participate in several inflammatory pathways [50]. Moreover, an interaction between macrophages and adipocytes has been reported [51], affecting insulin resistance mediated by the reduction of Akt phosphorylation, as we show here. Mesenchymal stromal cells, present in adipose tissue, are involved in anti-inflammatory processes, modulating the secretion of several interleukins [52]. We have found a reduction of vimentin, a mesenchymal marker [53], [54] in visceral fat depot, which could exacerbate inflammation and partial insulin resistance observed after chronic leptin treatment. In this regard, transplantation of adipose-derived mesenchymal cells increased phosphorylation of Akt [55], whereas macrophages inhibit differentiation of these cells. Hence, infiltration of macrophages in patients with obesity restrains this physiological process via secretion of proinflammatory cytokines [56]. Adipocytes also participate in the inflammatory state, expressing cytokines and their receptors [8]. Haptoglobin, a marker of inflammation is mainly synthetized by hepatocytes and adipocytes and its synthesis is upregulated by cytokines [57]. This adipose marker could exacerbate insulin resistance as it has been reported than impairment of glucose homeostasis is diminished by haptoglobin deficiency [58].

The data reported herein indicate that food restriction impairs insulin signal transduction in subcutaneous and visceral fat pads with leptin potentiating this effect in visceral tissue. Although reduction of food intake may initially attenuate insulin resistance [59], it has been stated that food restriction during the same length of time could lead to a down-regulation of insulin signaling in adipose tissue [60]. These authors demonstrate that this negative effect was due to the reduction in phosphorylation of the insulin receptor, together with a decrease of insulin receptor substrates, which could explain the reduced Akt phosphorylation. It appears that decreased insulin sensitivity in adipose tissue does not contribute in a significant manner to the change in the proinflammatory profile as pair-fed rats exhibit a cytokine profile similar to controls, with the exception of the levels of TNF-α. However, the pronounced reduction of Akt phosphorylation in visceral fat in the L group could be related to a direct effect of leptin and/or cytokines, as leptin directly decreases insulin-dependent autophosphorylation in adipocytes [13] and inflammatory cytokines may induce insulin resistance by decreasing insulin receptor levels [61], as we found in visceral fat. Alternatively, cytokine down-regulation of insulin receptor substrate expression [62] could explain the profound reduction of Akt phosphorylation reported here. Chronic leptin-treated rats present higher FOXO1 levels that could be the result of the lower Akt phosphorylation. In fact, activation of Akt reduces this factor by promoting subsequent polyubiquitylation and degradation by an ubiquitin proteasome system [63]. Thus, higher hypothalamic levels of FOXO1 in L group could be the result of a leptin-induced reduction in nuclear export of FOXO1 and cytoplasmic degradation [64]. Protein tyrosine phosphatase1B has been shown to be a negative regulator of insulin action and we have found an increase of PTP1B beside a reduction of insulin signaling in subcutaneous fat depot, as it has previously reported [65]. Unexpectedly, we observed a great reduction of levels of this protein in visceral fat. Nevertheless, it has been recently shown that PTP1B deficiency can exacerbate inflammatory processes [66], showing higher sensitivity to IFN-γ effects [67].

We cannot discard a negative effect of IFN-γ on insulin sensitivity in adipose tissue, as its administration induces sustained loss of insulin-stimulated glucose uptake, coincident with reduced Akt phosphorylation and STAT activation [61]. These authors also observed that JAK inhibition restored glucose uptake and Akt phosphorylation, in accordance with the results reported here showing an inverse relationship between IFN-γ levels and insulin signaling. In a similar way, increased serum IL-2 levels, as well as synthesis of IL-2 in epididymal fat, may contribute to reduce insulin sensitivity in adipose tissue [8]. Thus, these results suggest that leptin could affect insulin signaling in visceral tissue by modulating the synthesis of cytokines.

Several caveats should be taken into consideration when evaluating these results. Adipose tissue has different cell types and this study does not quantify the contribution of adipocytes or other cells in the generation of an inflammatory response. We also must take into account that we have reported a decrease in the content of epididymal fat, but not in subcutaneous adipose tissue, which was not been quantified. However, previous studies indicate that food restriction and leptin administration at similar doses decrease subcutaneous fat content by approximately 50% [68], [69]; thus, the contribution of fat depots to circulating cytokine levels is the resultant of the balance between cytokine synthesis and amount of adipose tissue. We cannot differentiate between the central effects of leptin from those due to increased circulating levels, as changes in metabolism and gene expression in fat are similar in most of the analyzed parameters by both administration routes [32] and denervation of white adipose tissue is necessary to make this distinction. In addition, synthesis of leptin in adipose tissue also may contribute to increased serum levels. Central infusion was chosen as it has more profound effects not only in food restriction and body weight, but also on carbohydrate and lipid metabolism [70], [71]. Finally, although we did not detect seizures or coma after i.p. insulin administration, the possible appearance of these symptoms in fasted animals should be taken into account. In addition, the fact that counterregulatory mechanisms in fasted animals will be activated in an attempt to avoid the hypoglycemia and this response could be different between the experimental groups must be considered. To avoid this marked decrease in serum glucose levels after the IPITT, a lower dose of insulin [72] could possibly be used.

In conclusion, our findings indicate that chronic leptin administration produces a serum inflammatory profile closely correlated with lower insulin sensitivity. Adipose tissue contributes to the generation of this adverse profile and the increased synthesis of cytokines seems to be related with the activation of leptin signaling and independent of insulin resistance in both fat depots. These results suggest that pharmacological inhibition of leptin signaling in adipose tissue could be of interest for reducing the low grade inflammatory state associated to obesity.

Materials and Methods

Materials

All chemicals were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) unless otherwise noted. The Inmmun-Star Western C Kit (ECL) was from Bio-Rad Laboratories (Hercules, CA, USA) and recombinant rat leptin was purchased from Preprotech (Rocky Hill, NJ, USA). Antibodies for phosphorylated (p)-Ser473-Akt, p-Ser727-signal transducer and activator of transcription factor 3 (pSer727-STAT3) and suppressor of cytokine signaling 3 (SOCS3) were from Cell Signaling Technology (Danvers, MA, USA), antibodies against Akt, F4/80, FOXO1 and IRβ were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), the antibody against STAT3 from R&D Systems (Minneapolis, MN, USA) and the antibody to actin from Thermo Fisher Scientific (Fremont, CA, USA). Antibody for PTP1B was from Millipore Corporate Headquarters (Billerica, MA, USA) and antibody against vimentin from Sigma-Aldrich. The corresponding secondary antibodies conjugated with horseradish-peroxidase were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

The rat leptin and insulin ELISA kits and multiplexed bead immunoassay for IL-2, -4, -6 -10, IFN-γ and TNF-α were from Millipore Corporate Headquarters (Billerica, MA, USA). TaqMan gene expression assays were purchased from Applied Biosystems (Foster City, CA, USA).

Animals

This study was approved by the Ethics Committee of the Universidad de Alcalá de Henares (SAF 2010–22277, Ministerio de Ciencia y Tecnología) and complied with Royal Decree 1201/2005 (Boletín Oficial del Estado, BOE no. 252) pertaining to the protection of experimental animals and with the European Communities Council Directive (86/609/EEC).

Thirty-six adult male Wistar rats (250±10 g) were individually caged with a 12-h light/dark cycle and fed standard chow and water ad libitum. After an overnight fast, rats were anesthetized (0.02 ml of ketamine/100 g wt and 0.04 ml of xylazine/100 g wt) and positioned in a stereotaxic apparatus. A cannula attached to an osmotic minipump (Alzet, Durect Corp., Cupertino, CA, USA) containing either saline or leptin was implanted into the right cerebral ventricle (−0.3 mm anteroposterior, 1.1 mm lateral from Bregma). Leptin was dissolved in saline plus 1% BSA and insulin was dissolved in PBS. Rats were treated icv for 14 days with either saline with 1% BSA or leptin (12 μg/day). To discriminate between the direct effects of leptin from those due to induction of decreased food intake, we included a pair-fed group that received the same amount of food consumed by the leptin-treated group the day before. Food intake and body weight were measured daily. Rats were sacrificed by decapitation at 8.00 h after a 12 h fast, inguinal fat as subcutaneous adipose tissue and epididymal fat as visceral tissue were isolated and blood collected.

Determination of insulin sensitivity, insulin tolerance and insulin resistance

On the last day of infusion, after a fasting period of 12 hours, 10 mIU of insulin or PBS in a volume of 5 μl were injected icv and rats were sacrificed by decapitation 2 hours later. This resulted in the following groups (n = 6 per group): chronic saline with 1% BSA (control, C), chronic saline with 1% BSA plus caloric restriction (pair-fed, PF), chronic leptin (leptin, L), chronic saline with 1% BSA plus acute insulin (insulin, I), 5) chronic saline with 1% BSA plus acute insulin and caloric restriction (insulin + pair-fed, PF+I) and 6) chronic leptin plus acute insulin (leptin + insulin, L+I). Blood was incubated at room temperature for 30 min, centrifuged at 1,500 g for 10 min at 4°C and serum collected and frozen at −80°C until determination of leptin, insulin and cytokines. Glycemia was measured before and 120 min after insulin administration via tail puncture (Accu-Check Sensor, Roche, Mannheim, Germany).

Insulin tolerance was assessed by performing an ip insulin tolerance test (IPITT) after the injection of a bolus of insulin (Regular Humuline, Lilly; 2 U/kg ip) and blood samples were drawn consecutively at 0, 30, 60, 90 and 120 min for glucose measurement [73], as described above.

In addition, homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated with the following formula: HOMA-IR = [Glucose (mmol/l) × insulin (μU/ml)]/22.5. Insulin sensitivity was measured as delta (Δ) in glycemia, calculated as glycemia after 120 min of insulin bolus minus glycemia before insulin bolus.

ELISAs

Serum leptin and insulin levels were measured with ELISA kits according to the manufacturer's instructions. The sensitivity of the assays for leptin and insulin were 0.04 and 0.2 ng/ml, respectively. The intra-assay variations were 2.2% for leptin and 1.9% for insulin and the inter-assay variations were 3.4% for leptin and 7.6% for insulin. Haptoglobin concentrations in adipose fat depots were determined by an ELISA kit from AssayPro (St. Charles, MO, USA). The intra- and inter-assay variations for haptoglobin were 5.2% and 7.9%, respectively.

Tissue homogenization and protein quantification

Adipose tissues were homogenized on ice in 500 μl radioimmunoprecipitation assay lysis buffer (RIPA; 50 mM NaH2PO4, 100 mM Na2HPO4, 0.1% sodium dodecyl sulfate, 0.5% NaCl, 1% Triton X-100) with EDTA-free protease inhibitors (Roche Diagnostics, Barcelona, Spain), 1 mM phenylmethanesulfonylfluoride and 5 mg/ml sodium deoxycholate for extraction of cytokines and leptin and insulin signaling-related proteins. The lysates were incubated overnight at −70°C and then centrifuged at 12,000 g for 5 min at 4°C. The supernatant was stored at −80°C until assayed. Total protein concentration was determined by the method of Bradford (Bio-Rad Laboratories).

Multiplexed bead immunoassay

Serum and tissue IL-2, -4, -6, -10, IFN-γ and TNF-α concentrations were measured by a multiplexed bead immunoassay. Briefly, after blockage of the filter plate with assay buffer, wells were washed by using a vacuum manifold and beads with different fluorescent labeling for each antigen conjugated to the appropriate antibodies and serum (25 μl each) were added and then incubated for 18 hours at 4°C. Wells were washed and 25 μl of antibody conjugated to biotin were added. After incubation for 30 min at room temperature, beads were incubated during 30 min with 50 μl streptavidin conjugated to phycoerythrin. After washing, beads were resuspended and a minimum of 50 beads per parameter was analyzed in the Bio-Plex suspension array system 200 (Bio-Rad Laboratories, Madrid, Spain). Raw data (mean fluorescence intensity, MFI) was analyzed by using the Bio-Plex Manager Software 4.1 (Bio-Rad Laboratories). Sensitivity is approximately 2–5 pg/ml, mean intra-assay variation was 8.0% and mean inter-assay variation was 12.6% for all cytokines.

Western blotting

Western blotting was used to determine levels of SOCS3 and insulin receptor and activation of STAT3 and Akt in subcutaneous and visceral adipose tissues. The proteins were resolved on a 10% SDS-polyacrylamide gel and then transferred to polyvinyl difluoride (PVDF) membranes. Membranes were blocked with TTBS containing 5% (w/v) BSA during 2 h at 25°C and incubated with the corresponding primary antibody (diluted 1:1000) in TTBS at 4°C overnight. The membranes were subsequently washed and incubated with the corresponding secondary antibody conjugated with peroxidase at a dilution of 1:2000 in TTBS during 90 min at 25°C. The proteins were detected by chemiluminescence with an ECL system. Quantification of the bands obtained was carried out by densitometry using a Kodak Gel Logic 1500 Image Analysis system and Molecular Imaging Software version 4.0 (Rochester, NY, USA). Insulin receptor, FOXO1, PTP1B, SOCS3 and vimentin were normalized with actin and pAkt and pSer727STAT3 with their total forms.

RNA purification and real time PCR analysis

Total RNA was extracted according to the Qiazol protocol (Qiagen Sciences, Maryland, USA). Reverse transcription was performed on 2 μg of total RNA using the high-capacity cDNA archive kit (Applied Biosystems). Real-time PCR was performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using TaqMan PCR Master Mix (Applied Biosystems) and the thermocycler parameters recommended by the manufacturer. PCRs were performed in duplicate in a total volume of 50 μl, containing 25 μl of the reverse transcription reaction. TaqMan gene expression assays were used for IL-2, IL-4, IFN-γ, leptin, ObR and TNF-α (Rn00587673_m1, Rn01456866_m1, Rn00594078_m1, Rn00565158_m1, Rn01433205_m1 and Rn01525859_g1, respectively; Applied Biosystems). All expression assays were performed following the manufacturer's procedures, except ObR, that was done following the modifications of Siegrist-Kaiser et al. [74]. Relative gene expression comparisons were carried-out using an invariant endogenous control (actin). According to manufacturer's guidelines, the ΔΔCT method was used for relative quantification.

Statistical analysis

Differences between groups were analyzed by a one-way ANOVA followed by a Bonferronís test. Statistical significance was set at p<0.05. Pearson's correlation coefficient r was used to measure the degree of association between different variables in each group. Two-tailed p values <0.05 were considered significant. These correlations were conducted with Prism software 4.00 (GraphPad, San Diego, CA, USA). The trapezoidal rule of GraphPad Prism was employed to calculate the area under the curve. Data are expressed as mean ± SEM.

Acknowledgments

The authors wish to thank Dr. Julie A. Chowen, Dr. Laura M. Frago and Dr. Gabriel A. Martos-Moreno for the critical review of the manuscript, Francisca Díaz for the excellent technical support and Luis Miguel Jiménez from Deltaclón for the generous gift of the multiplexed bead immunoassay kits.

Author Contributions

Conceived and designed the experiments: EBR EAF VB. Performed the experiments: EBR SC APC. Analyzed the data: EBR VB. Contributed reagents/materials/analysis tools: EAF JA VB. Wrote the paper: JA VB. Supervised the study: VB.

References

  1. 1. Wong S, Pinkney J (2004) Role of cytokines in regulating feeding behaviour. Curr Drug Targets 5: 251–263. doi: 10.2174/1389450043490532
  2. 2. Tapia-González S, García-Segura LM, Tena-Sempere M, Frago LM, Castellano JM, et al. (2011) Activation of microglia in specific hypothalamic nuclei and the cerebellum of adult rats exposed to neonatal overnutrition. J Neuroendocrinol 23: 365–370. doi: 10.1111/j.1365-2826.2011.02113.x
  3. 3. Martos-Moreno GA, Burgos-Ramos E, Canelles S, Argente J, Barrios V (2010) Evaluation of a multiplex assay for adipokine concentrations in obese children. Clin Chem Lab Med 48: 1439–1446. doi: 10.1515/cclm.2010.276
  4. 4. Wang Y, Rimm EB, Stampfer MJ, Willett WC, Hu FB (2005) Comparison of abdominal adiposity and overall obesity in predicting risk of type 2 diabetes among men. Am J Clin Nutr 81: 555–563.
  5. 5. Thaler JP, Schwartz MW (2010) Minireview: Inflammation and obesity pathogenesis: the hypothalamus heats up. Endocrinology 151: 4109–4115. doi: 10.1210/en.2010-0336
  6. 6. Stofkova A (2009) Leptin and adiponectin: from energy and metabolic dysbalance to inflammation and autoimmunity. Endocr Regul 43: 157–168.
  7. 7. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, et al. (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112: 1796–1808. doi: 10.1172/jci19246
  8. 8. Meijer K, de Vries M, Al-Lahham S, Bruinenberg M, Weening D, et al. (2011) Human primary adipocytes exhibit immune cell function: adipocytes prime inflammation independent of macrophages. PLoS One 6: e17154. doi: 10.1371/journal.pone.0017154
  9. 9. Tanaka M, Suganami T, Kim-Saijo M, Toda C, Tsuiji M, et al. (2011) Role of central leptin signaling in the starvation-induced alteration of B-cell development. J Neurosci 31: 8373–8380. doi: 10.1523/jneurosci.6562-10.2011
  10. 10. Kiguchi N, Maeda T, Kobayashi Y, Fukazawa Y, Kishioka S (2009) Leptin enhances CC-chemokine ligand expression in cultured murine macrophage. Biochem Biophys Res Commun 384: 311–315. doi: 10.1016/j.bbrc.2009.04.121
  11. 11. Paz-Filho G, Esposito K, Hurwitz B, Sharma A, Dong C, et al. (2008) Changes in insulin sensitivity during leptin replacement therapy in leptin-deficient patients. Am J Physiol Endocrinol Metab 295: E1401–E1408. doi: 10.1152/ajpendo.90450.2008
  12. 12. Burgos-Ramos E, Chowen JA, Arilla-Ferreiro E, Canelles S, Argente J, et al. (2011) Chronic central leptin infusion modifies the response to acute central insulin injection by reducing the interaction of the insulin receptor with IRS2 and increasing its association with SOCS3. J Neurochem 117: 175–185. doi: 10.1111/j.1471-4159.2011.07191.x
  13. 13. Pérez C, Fernández-Galaz C, Fernández-Agulló T, Arribas C, Andrés A, et al. (2004) Leptin impairs insulin signaling in rat adipocytes. Diabetes 53: 347–353. doi: 10.2337/diabetes.53.2.347
  14. 14. Lionetti L, Mollica MP, Lombardi A, Cavaliere G, Gifuni G, et al. (2009) From chronic overnutrition to insulin resistance: the role of fat-storing capacity and inflammation. Nutr Metab Cardiovasc Dis 19: 146–152. doi: 10.1016/j.numecd.2008.10.010
  15. 15. Xi L, Qian Z, Xu G, Zhou C, Sun S (2007) Crocetin attenuates palmitate-induced insulin insensitivity and disordered tumor necrosis factor-alpha and adiponectin expression in rat adipocytes. Br J Pharmacol 151: 610–617. doi: 10.1038/sj.bjp.0707276
  16. 16. Sloan C, Tuinei J, Nemetz K, Frandsen J, Soto J, et al. (2011) Central leptin signaling is required to normalize myocardial fatty acid oxidation rates in caloric-restricted ob/ob mice. Diabetes 60: 1424–1434. doi: 10.2337/db10-1106
  17. 17. Escrivá F, Gavete ML, Fermín Y, Pérez C, Gallardo N, et al. (2007) Effect of age and moderate food restriction on insulin sensitivity in Wistar rats: role of adiposity. J Endocrinol 194: 131–141. doi: 10.1677/joe.1.07043
  18. 18. Dalmas E, Rouault C, Abdennour M, Rovere C, Rizkalla S, et al. (2011) Variations in circulating inflammatory factors are related to changes in calorie and carbohydrate intakes early in the course of surgery-induced weight reduction. Am J Clin Nutr 94: 450–458. doi: 10.3945/ajcn.111.013771
  19. 19. Bastard JP, Maachi M, Lagathu C, Kim MJ, Caron M, et al. (2006) Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw 17: 4–12.
  20. 20. Marino JS, Xu Y, Hill JW (2011) Central insulin and leptin-mediated autonomic control of glucose homeostasis. Trends Endocrinol Metab 22: 275–285. doi: 10.1016/j.tem.2011.03.001
  21. 21. Canavan B, Salem RO, Schurgin S, Koutkia P, Lipinska I, et al. (2005) Effects of physiological leptin administration on markers of inflammation, platelet activation, and platelet aggregation during caloric deprivation. J Clin Endocrinol Metab 90: 5779–5785. doi: 10.1210/jc.2005-0780
  22. 22. Aygun AD, Gungor S, Ustundag B, Gurgoze MK, Sen Y (2005) Proinflammatory cytokines and leptin are increased in serum of prepubertal obese children. Mediators Inflamm 2005: 180–183. doi: 10.1155/mi.2005.180
  23. 23. Lord GM, Matarese G, Howard JK, Bloom SR, Lechler RI (2002) Leptin inhibits the anti-CD3-driven proliferation of peripheral blood T cells but enhances the production of proinflammatory cytokines. J Leukoc Biol 72: 330–338.
  24. 24. Lo HC, Lin SC, Wang YM (2004) The relationship among serum cytokines, chemokine, nitric oxide, and leptin in children with type 1 diabetes mellitus. Clin Biochem 37: 666–672. doi: 10.1016/j.clinbiochem.2004.02.002
  25. 25. Carpagnano GE, Spanevello A, Curci C, Salerno F, Palladino GP, et al. (2007) IL-2, TNF-alpha, and leptin: local versus systemic concentrations in NSCLC patients. Oncol Res 16: 375–381.
  26. 26. Han SN, Jeon KJ, Kim MS, Kim HK, Lee AJ (2011) Obesity with a body mass index under 30 does not significantly impair the immune response in young adults. Nutr Res 31: 362–369. doi: 10.1016/j.nutres.2011.04.002
  27. 27. Mraz M, Lacinova Z, Drapalova J, Haluzikova D, Horinek A, et al. (2011) The effect of very-low-calorie diet on mRNA expression of inflammation-related genes in subcutaneous adipose tissue and peripheral monocytes of obese patients with type 2 diabetes mellitus. J Clin Endocrinol Metab 96: E606–E613. doi: 10.1210/jc.2010-1858
  28. 28. Morrison CD, Daniel JA, Holmberg BJ, Djiane J, Raver N, et al. (2001) Central infusion of leptin into well-fed and undernourished ewe lambs: effects on feed intake and serum concentrations of growth hormone and luteinizing hormone. J Endocrinol 168: 317–324. doi: 10.1677/joe.0.1680317
  29. 29. Cusin I, Rouru J, Visser T, Burger AG, Rohner-Jeanrenaud F (2000) Involvement of thyroid hormones in the effect of intracerebroventricular leptin infusion on uncoupling protein-3 expression in rat muscle. Diabetes 49: 1101–1105. doi: 10.2337/diabetes.49.7.1101
  30. 30. Cettour-Rose P, Burger AG, Meier CA, Visser TJ, Rohner-Jeanrenaud F (2002) Central stimulatory effect of leptin on T3 production is mediated by brown adipose tissue type II deiodinase. Am J Physiol Endocrinol Metab 283: E980–E987.
  31. 31. Ramsay TG, Richards MP (2004) Hormonal regulation of leptin and leptin receptor expression in porcine subcutaneous adipose tissue. J Anim Sci 82: 3486–3492.
  32. 32. Yoshida T, Monkawa T, Hayashi M, Saruta T (1997) Regulation of expression of leptin mRNA and secretion of leptin by thyroid hormone in 3T3-L1 adipocytes. Biochem Biophys Res Commun 232: 822–826. doi: 10.1006/bbrc.1997.6378
  33. 33. Prieur X, Tung YC, Griffin JL, Farooqi IS, O'Rahilly S, et al. (2008) Leptin regulates peripheral lipid metabolism primarily through central effects on food intake. Endocrinology 149: 5432–5439. doi: 10.1210/en.2008-0498
  34. 34. Reidy SP, Weber J (2000) Leptin: an essential regulator of lipid metabolism. Comp Biochem Physiol A Mol Integr Physiol 125: 285–298. doi: 10.1016/s1095-6433(00)00159-8
  35. 35. Moschen AR, Molnar C, Enrich B, Geiger S, Ebenbichler CF, et al. (2011) Adipose and liver expression of interleukin (IL)-1 family members in morbid obesity and effects of weight loss. Mol Med 17: 840–845.
  36. 36. Moschen AR, Molnar C, Geiger S, Graziadei I, Ebenbichler CF, et al. (2010) Anti-inflammatory effects of excessive weight loss: potent suppression of adipose interleukin 6 and tumour necrosis factor alpha expression. Gut 59: 1259–1264. doi: 10.1136/gut.2010.214577
  37. 37. Roth CL, Kratz M, Ralston MM, Reinehr T (2011) Changes in adipose-derived inflammatory cytokines and chemokines after successful lifestyle intervention in obese children. Metabolism 60: 445–452. doi: 10.1016/j.metabol.2010.03.023
  38. 38. Nakhjavani M, Esteghamati A, Tarafdari AM, Nikzamir A, Ashraf H, et al. (2011) Association of plasma leptin levels and insulin resistance in diabetic women: a cross-sectional analysis in an Iranian population with different results in men and women. Gynecol Endocrinol 27: 14–19. doi: 10.3109/09513590.2010.487583
  39. 39. Kalra SP (2009) Central leptin gene therapy ameliorates diabetes type 1 and 2 through two independent hypothalamic relays; a benefit beyond weight and appetite regulation. Peptides 30: 1957–1963. doi: 10.1016/j.peptides.2009.07.021
  40. 40. Yang YY, Tsai TH, Huang YT, Lee TY, Chan CC, et al. (2012) Hepatic endothelin-1 and endocannabinoids-dependent effects of hyperleptinaemia in nonalcoholic steatohepatitis-cirrhotic rats. Hepatology 55: 1540–1550. doi: 10.1002/hep.25534
  41. 41. Han Y, Joe Y, Seo E, Lee SR, Park MK, et al. (2010) The hyperleptinemia and ObRb expression in hyperphagic obese rats. Biochem Biophys Res Commun 394: 70–74. doi: 10.1016/j.bbrc.2010.02.104
  42. 42. Zha JM, Di WJ, Zhu T, Xie Y, Yu J, et al. (2009) Comparison of gene transcription between subcutaneous and visceral adipose tissue in Chinese adults. Endocr J 56: 935–944. doi: 10.1507/endocrj.k09e-091
  43. 43. Lefebvre AM, Laville M, Vega N, Riou JP, van Gaal L, et al. (1998) Depot-specific differences in adipose tissue gene expression in lean and obese subjects. Diabetes 47: 98–103. doi: 10.2337/diabetes.47.1.98
  44. 44. Maya-Monteiro CM, Almeida PE, D'Avila H, Martins AS, Rezende AP, et al. (2008) Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism. J Biol Chem 283: 2203–2210. doi: 10.1074/jbc.m706706200
  45. 45. Fernández-Riejos P, Najib S, Santos-Alvarez J, Martín-Romero C, Pérez-Pérez A, et al. (2010) Role of leptin in the activation of immune cells. Mediators Inflamm 2010: 568343. doi: 10.1155/2010/568343
  46. 46. Neels JG, Olefsky JM (2006) Inflamed fat: what starts the fire? J Clin Invest 116: 33–35. doi: 10.1172/jci27280
  47. 47. Zeyda M, Stulnig TM (2007) Adipose tissue macrophages. Immunol Lett 112: 61–67. doi: 10.1016/j.imlet.2007.07.003
  48. 48. Gove ME, Rhodes DH, Pini M, van Baal JW, Sennello JA, et al. (2009) Role of leptin receptor-induced STAT3 signaling in modulation of intestinal and hepatic inflammation in mice. J Leukoc Biol 85: 491–496. doi: 10.1189/jlb.0808508
  49. 49. Barzilai N, Wang J, Massilon D, Vuguin P, Hawkins M, et al. (1997) Leptin selectively decreases visceral adiposity and enhances insulin action. J Clin Invest 100: 3105–3110. doi: 10.1172/jci119865
  50. 50. Mayi TH, Daoudi M, Derudas B, Gross B, Bories G, et al.. (2012) Human adipose tissue macrophages display activation of cancer-related pathways. J Biol Chem (doi: 10.1074/jbc.M111.315200).
  51. 51. Xie L, Ortega MT, Mora S, Chapes SK (2010) Interactive changes between macrophages and adipocytes. Clin Vaccine Immunol 17: 651–659. doi: 10.1128/cvi.00494-09
  52. 52. Raicevic G, Najar M, Stamatopoulos B, De Bruyn C, Meuleman N, et al. (2011) The source of human mesenchymal stromal cells influences their TLR profile as well as their functional properties. Cell Immunol 270: 207–216. doi: 10.1016/j.cellimm.2011.05.010
  53. 53. Wenceslau CV, Miglino MA, Martins DS, Ambrósio CE, Lizier NF, et al. (2011) Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow. Tissue Eng Part A 17: 2165–2176. doi: 10.1089/ten.tea.2010.0678
  54. 54. Shi JG, Fu WJ, Wang XX, Xu YD, Li G, et al.. (2012) Transdifferentiation of human adipose-derived stem cells into urothelial cells: potential for urinary tract tissue engineering. Cell Tissue Res (doi: 10.1007/s00441-011-1317-0).
  55. 55. Da Justa Pinheiro CH, de Queiroz JC, Guimarães-Ferreira L, Vitzel KF, Nachbar RT, et al.. (2011) Local Injections of Adipose-Derived Mesenchymal Stem Cells Modulate Inflammation and Increase Angiogenesis Ameliorating the Dystrophic Phenotype in Dystrophin-Deficient Skeletal Muscle. Stem Cell Rev (doi: 10.1007/s12015-011-9304-0).
  56. 56. Bilkovski R, Schulte DM, Oberhauser F, Mauer J, Hampel B, et al. (2011) Adipose tissue macrophages inhibit adipogenesis of mesenchymal precursor cells via wnt-5a in humans. Int J Obes (Lond) 35: 1450–1454. doi: 10.1038/ijo.2011.6
  57. 57. Do Nascimento CO, Hunter L, Trayhurn P (2004) Regulation of haptoglobin gene expression in 3T3-L1 adipocytes by cytokines, catecholamines, and PPARgamma. Biochem Biophys Res Commun 313: 702–708. doi: 10.1016/j.bbrc.2003.12.008
  58. 58. Lisi S, Gamucci O, Vottari T, Scabia G, Funicello M, et al. (2011) Obesity-associated hepatosteatosis and impairment of glucose homeostasis are attenuated by haptoglobin deficiency. Diabetes 60: 2496–2505. doi: 10.2337/db10-1536
  59. 59. Rodrigues L, Crisóstomo J, Matafome P, Louro T, Nunes E, et al. (2011) Dietary restriction improves systemic and muscular oxidative stress in type 2 diabetic Goto-Kakizaki rats. J Physiol Biochem 67: 613–619. doi: 10.1007/s13105-011-0108-0
  60. 60. Alonso A, Fernández Y, Fernández R, Ordóñez P, Moreno M, et al. (2005) Effect of food restriction on the insulin signalling pathway in rat skeletal muscle and adipose tissue. J Nutr Biochem 16: 602–609. doi: 10.1016/j.jnutbio.2005.03.002
  61. 61. McGillicuddy FC, Chiquoine EH, Hinkle CC, Kim RJ, Shah R, et al. (2009) Interferon gamma attenuates insulin signaling, lipid storage, and differentiation in human adipocytes via activation of the JAK/STAT pathway. J Biol Chem 284: 31936–31944. doi: 10.1074/jbc.m109.061655
  62. 62. Jager J, Grémeaux T, Cormont M, Le Marchand-Brustel Y, Tanti JF (2007) Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression. Endocrinology 148: 241–251. doi: 10.1210/en.2006-0692
  63. 63. Matsuzaki H, Daitoku H, Hatta M, Tanaka K, Fukamizu A (2003) Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation. Proc Natl Acad Sci USA 100: 11285–11290. doi: 10.1073/pnas.1934283100
  64. 64. Fukuda M, Jones JE, Olson D, Hill J, Lee CE, et al. (2008) Monitoring FoxO1 localization in chemically identified neurons. J Neurosci 28: 13640–13648. doi: 10.1523/jneurosci.4023-08.2008
  65. 65. Rondinone CM, Trevillyan JM, Clampit J, Gum RJ, Berg C, et al. (2002) Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis. Diabetes 51: 2405–2411. doi: 10.2337/diabetes.51.8.2405
  66. 66. Berdnikovs S, Pavlov VI, Abdala-Valencia H, McCary CA, Klumpp DJ, et al. (2012) PTP1B deficiency exacerbates inflammation and accelerates leukocyte trafficking in vivo. J Immunol 188: 874–884. doi: 10.4049/jimmunol.1004108
  67. 67. Heinonen KM, Bourdeau A, Doody KM, Tremblay ML (2009) Protein tyrosine phosphatases PTP-1B and TC-PTP play nonredundant roles in macrophage development and IFN-gamma signaling. Proc Natl Acad Sci USA 106: 9368–9372. doi: 10.1073/pnas.0812109106
  68. 68. Dugdale AH, Curtis GC, Cripps P, Harris PA, Argo CM (2010) Effect of dietary restriction on body condition, composition and welfare of overweight and obese pony mares. Equine Vet J 42: 600–610. doi: 10.1111/j.2042-3306.2010.00110.x
  69. 69. Pardo M, Roca-Rivada A, Al-Massadi O, Seoane LM, Camiña JP, et al. (2010) Peripheral leptin and ghrelin receptors are regulated in a tissue-specific manner in activity-based anorexia. Peptides 31: 1912–1919. doi: 10.1016/j.peptides.2010.06.022
  70. 70. Ramsey JJ, Kemnitz JW, Colman RJ, Cunningham D, Swick AG (1998) Different central and peripheral responses to leptin in rhesus monkeys: brain transport may be limited. J Clin Endocrinol Metab 83: 3230–3235. doi: 10.1210/jcem.83.9.5073
  71. 71. Penn DM, Jordan LC, Kelso EW, Davenport JE, Harris RB (2006) Effects of central or peripheral leptin administration on norepinephrine turnover in defined fat depots. Am J Physiol Regul Integr Comp Physiol 291: R1613–R1621. doi: 10.1152/ajpregu.00368.2006
  72. 72. Malakauskas SM, Kourany WM, Zhang XY, Lu D, Stevens RD, et al. (2009) Increased insulin sensitivity in mice lacking collectrin, a downstream target of HNF-1alpha. Mol Endocrinol 23: 881–892. doi: 10.1210/me.2008-0274
  73. 73. Ndisang JF, Lane N, Syed N, Jadhav A (2010) Up-regulating the heme oxygenase system with hemin improves insulin sensitivity and glucose metabolism in adult spontaneously hypertensive rats. Endocrinology 151: 549–560. doi: 10.1210/en.2009-0471
  74. 74. Siegrist-Kaiser CA, Pauli V, Juge-Aubry CE, Boss O, Pernin A, et al. (1997) Direct effects of leptin on brown and white adipose tissue. J Clin Invest 100: 2858–2864. doi: 10.1172/jci119834