JNK and IKKβ phosphorylation is reduced by glucocorticoids in adipose tissue from insulin-resistant rats
Introduction
Glucocorticoids (GCs) induce IR (insulin resistance) in peripheral tissues. In the adipose tissue, GCs increase lipolysis, while in the skeletal muscles and liver, GCs decrease the glucose uptake and increase the gluconeogenesis [1], [2]. These diabetogenic effects of GCs are exerted, at least in part, by antagonizing insulin actions [3], [4]. Insulin signaling is initiated upon insulin binding to its membrane receptor and the subsequent activation of intrinsic tyrosine kinase autophosphorylation that then leads to the phosphorylation of insulin receptor substrate (IRS)-1. The phosphorylated IRS-1 then activates the phosphatidylinositol 3-kinase (Pi3-kinase). Pi3-kinase seems to be the major mediator of protein kinase B (PKB) activation, which is essential for most of the physiological responses to insulin [5], [6], [7]. To become completely activated, PKB requires phosphorylation on two key residues, one on serine 473 and other on threonine 308 [8], [9]. These events result in glycogen synthesis and in the translocation of vesicles containing glucose transporter (GLUT)-4 to the cellular membrane for glucose uptake by skeletal muscles and fat cells [10], [11].
Proinflammatory proteins decrease insulin sensitivity by impairing the phosphorylation of proteins that participate in insulin signaling such as IRS-1 and PKB [12]. During obesity conditions, such as in metabolic syndrome (MS), there is an elevation in circulating proinflammatory cytokines, such as interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF), a process known as low-grade inflammation [13]. These cytokines may bind to specific receptors that result in the activation of protein kinases, including c-Jun-N-terminal kinase (JNK) and an inhibitor of the nuclear factor kappa-B (IKKβ) [14], [15]. These protein kinases activate the gene transcription of nuclear factor kappa B (NFκB) and activator protein 1 (AP1) [16], [17] and may also interfere negatively with insulin signaling by phosphorylating IRS-1 and decreasing PKB phosphorylation [18], [19]. In parallel, the released saturated fatty acids (SFA) during obesity may also bind to the receptors that trigger intracellular inflammatory signaling and may lead to subsequent impairment in insulin signaling [20]. This attenuation in insulin signaling is also associated with conditions of GC excess [3], [21]. Treatment with dexamethasone for up to 11 consecutive days promotes a reduction in PKB phosphorylation in the skeletal muscles [3] and decreases IRS-1 and PKB phosphorylation in the white adipose tissue of rats [21]. However, the mechanisms underlying GC-induced impairment in insulin signaling are not yet fully elucidated.
GCs exert their actions by binding to the glucocorticoid receptor (GR), which belongs to the nuclear receptor family, and act as the transcription factors [22], [23]. GR attenuates cellular inflammatory responses by suppressing the gene transcription of certain inflammatory components [24]. This GC-induced gene suppression occurs through the direct interaction of the GC–GR complex at glucocorticoid-responsive elements (GREs) and/or by protein–protein interactions of the GC–GR complex with NFkB or AP-1 signals [25], [26], [27]. When in excess, GCs may promote the downregulation of GR, as demonstrated after both in vitro and in vivo GC exposures [28]. However, whether adipose tissue is subjected to alleviated suppression of proinflammatory signaling concomitant with GR downregulation remains to be elucidated.
Dexamethasone-based therapies are routinely prescribed in clinical practice to treat several diseases based in their anti-inflammatory and immunosuppressive actions [29], [30]. Considering that the patients submitted to GC therapies develop peripheral IR and that there is an evidence that the GC exposure may lead to GR downregulation, we aimed to investigate whether there are any associations between insulin signaling attenuation and an alteration in proinflammatory signaling in the adipose tissue from GC-treated rats. We demonstrated that 5 days of dexamethasone administration in rats induced peripheral IR. These rats exhibited decreased PKB and increased IRS-1 phosphorylation at serine residues without increasing JNK and IKKβ phosphorylation in epididymal adipose tissue.
Section snippets
Ethical approval
The experiments with rats were approved by the Federal University of Santa Catarina Committee for Ethics in Animal Experimentation (approval ID: PP00782).
Materials
Dexamethasone phosphate (Decadron®) was purchased from Aché (Campinas, SP, Brazil). Human recombinant insulin (Humulin®) was from Lilly (Indianapolis, IN, USA). The reagents used in the glucose tolerance test, pyruvate tolerance test, and lipolysis and hepatic glycogen protocols were from LabSynth (Diadema, SP, Brazil) and Sigma (St. Louis,
Characteristics of the rats
There were no differences in the body weight between the CTL and DEX groups before the beginning of the treatment (Fig. 1A). The body weights were significantly reduced in the DEX rats after 48 h of dexamethasone administration and remained with a linear and sustained reduction until euthanasia. On the day of euthanasia, the body weight in the DEX group was 17% lower than that in the CTL group. The final body weights of the groups were 332 ± 11 and 275 ± 6 g for the CTL and for the DEX rats,
Discussion
In this study, we reproduced the well-established side effects of GC excess: reduced body weight and food intake [3], [21], [33], increased blood glucose levels, increased plasma insulin and triacylglycerol levels [3], [21], [33], [34], [46], increased hepatic glycogen content [33], [34], and reduced insulin sensitivity and glucose tolerance [21], [33], [34], [46]. In agreement with previous studies [3], [21], dexamethasone treatment resulted in the attenuation of insulin signaling in the
Authorship contributions
Participated in research design: Katia Motta and Alex Rafacho. Conducted experiments: Katia Motta, Amanda Marreiro Barbosa and Franciane Bobinski. Contributed analytic tools and data analysis: Katia Motta and Alex Rafacho. Performed data collection and interpretation: Katia Motta and Alex Rafacho. Contributed to manuscript writing: Katia Motta, Antonio Carlos Boschero and Alex Rafacho.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This study was supported by grants from the CNPq (ID number 471397/2011-3). The authors appreciate the technical assistance of Cristiane dos Santos, Francielle BD Ferreira and Luiz Gonçalves-Neto and the kind donation of the ELISA kits by Dr. Adair R S Santos.
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