Role of extracellular signal-regulated kinase 5 in adipocyte signaling

Abstract

Increased adiposity due to energy imbalance is a critical factor of the epidemic crisis of obesity and type II diabetes. In addition to the obvious role in energy storage, regulatory factors are secreted from adipose depots to control appetite and cellular homeostasis. Complex signaling cross-talks within adipocyte are also evident due to the metabolic and immune nature of adipose depots. Here, we uncover a role of extracellular signal-regulated kinase 5 (ERK5) in adipocyte signaling. We find that deletion of ERK5 in adipose depots (adipo-ERK5(-/-)) increases adiposity, in part, due to increased food intake. Dysregulated secretion of adipokines, leptin resistance, and impaired glucose handling are also found in adipo-ERK5(-/-) mice. Mechanistically, we show that ERK5 impinges on transcription factor NFATc4. Decreased phosphorylation at the conserved gate-keeping Ser residues and increased nuclear localization of NFATc4 are found in adipo-ERK5(-/-) mice. We also find attenuated PKA activation in adipo-ERK5(-/-) mice. In response to stimulation of β-adrenergic G-protein-coupled receptor, we find decreased NFATc4 phosphorylation and impaired PKA activation in adipo-ERK5(-/-) mice. Reduced cAMP accumulation and increased phosphodiesterase activity are also found. Together, these results demonstrate integration of ERK5 with NFATc4 nucleo-cytoplasmic shuttling and PKA activation in adipocyte signaling.

Keywords: Adipose tissue; MAP Kinases (MAPKs); Metabolic Regulation; NFAT Transcription Factor; Protein Kinase A (PKA).

FIGURE 1.

Reduced expression of ERK5 in adipo-ERK5^−/− mice. A, expression of ERK5 protein in epididymal fat pads (WAT), heart, liver, and skeletal muscle of adipo-ERK5^−/− and control mice. B, expression of ERK5 and actin in epididymal fat pads of adipo-ERK5^−/− and control mice was quantitated by RT-quantitative PCR. Relative expression of ERK5 and actin was normalized to the expression of cyclophilin and shown. C, detection of ERK5 expression in adipose depots of adipo-ERK5^−/− and control mice by immunohistochemistry. *, crownlike morphology of adipocytes in adipo-ERK5^−/− mice. D, adipocytes and stromal vascular cells were separated by density centrifugation. The levels of ERK5 in the isolated adipocytes and stromal vascular cells were determined. *, p < 0.05. Error bars, S.E.

FIGURE 2.

Increased adiposity in adipo-ERK5^−/− mice. A and B, adipo-ERK5^−/− and control mice were fed ad libitum with regular chow (A) or high fat diet (B). Body weight of the mice was determined for 20 weeks and shown. C, 12-week-old adipo-ERK5^−/− and control mice were subjected to EchoMRI to determine fat mass and lean mass. *, p < 0.05; #, p < 0.005. Error bars, S.E.

FIGURE 3.

Altered metabolic parameters in adipo-ERK5^−/− mice. Oxygen consumption (VO₂) (A), carbon dioxide production (VCO₂) (B), respiratory quotient (RER) (C and D), energy expenditure (Heat) (E), food intake (F), meal number (G), and meal size (H) of 12-week-old adipo-ERK5^−/− and control mice (n = 8) were determined by an indirect open circuit calorimeter system. Locomotor activity (I and J) of the adipo-ERK5^−/− and control mice were also shown. *, p < 0.05; #, p < 0.005. Error bars, S.E.

FIGURE 4.

Changes in adipokine levels in adipo-ERK5^−/− mice. The levels of adiponectin, leptin, resistin, and PAI-1 in serum of 20-year-old (A) and 12-year-old (B) adipo-ERK5^−/− and control mice (n = 4–6) were determined by mouse adipokine multiplex assays. Fasting levels of glucose and insulin in adipo-ERK5^−/− and control mice were also shown. Effects of a high fat diet (HFD) on adipokine levels were also determined. *, p < 0.05; #, p < 0.005. Error bars, S.E.

FIGURE 5.

Impaired glucose handling and reduced insulin sensitivity in adipo-ERK5^−/− mice. Eight-week-old adipo-ERK5^−/− (n = 7) and control mice (n = 9) were subjected to glucose tolerance tests (A) and insulin tolerance tests (B). The glucose levels were measured at the indicated times and presented. The area under the curve was also determined. *, p < 0.05; #, p < 0.005. Error bars, S.E.

FIGURE 6.

Leptin resistance in adipo-ERK5^−/− mice. A, recombinant murine leptin (2 mg/kg) or saline was administered to overnight-fasted, 12-week-old adipo-ERK5^−/− and control mice via intraperitoneal injection. Levels of phospo-STAT3 were determined by immunoprecipitations (IP) and immunoblottings (IB). Levels of STAT3 were also shown. B, relative level of STAT3 phosphorylation was determined by densitometry and shown. *, p < 0.05; NS, not significant.

FIGURE 7.

Integration of ERK5 and NFATc4 in adipocyte signaling. A, tissue extracts from epididymal fat pads of 8-week-old adipo-ERK5^−/− and control mice were examined to determine the levels of NFATc4 phosphorylation. B, WAT isolated from adipo-ERK5^−/− and control mice (n = 3) were challenged with β-adrenergic receptor agonist isoproterenol (Iso; 1 μm) for the times indicated. Tissue extracts prepared were used to determine the levels of NFATc4 phosphorylation. C, paraffin sections of epididymal fat pads of 8-week-old adipo-ERK5^−/− and control mice were stained with rabbit polyclonal NFATc4 antibody (green). DNA in nuclei was visualized using DAPI and represented in the red channel. The presence or the lack of NFATc4 in the nucleus is indicated by arrowheads. Error bars, S.E.

FIGURE 8.

Integration of ERK5 and PKA activation in adipocyte signaling. A, tissue extracts from epididymal fat pads of 8-week-old adipo-ERK5^−/− and control mice were examined to determine the levels of activation of PKA by immunoblotting analysis using phospho-PKA motif antibodies (Phospho-PKA substrates). B and C, WAT isolated from adipo-ERK5^−/− and control mice (n = 3) were challenged with β-adrenergic receptor agonist isoproterenol (Iso; 1 μm) (B) or CL316,243 (CL; 1 μm) (C) for the times indicated. Tissue extracts prepared were used to determine activation of PKA by immunoblotting analysis using phospho-PKA motif antibodies (Phospho-PKA substrates). The effect of isoproterenol stimulation on phosphorylation of hormone-sensitive lipase (HSL) was also shown (B). D, primary embryonic fibroblasts (MEFs) were challenged with β-adrenergic receptor agonist isoproterenol (10 μm) for the times indicated, in the absence (DMSO) and presence (BIX2189) of MEK5 inhibitor (BIX2189; 10 μm). Activation of PKA was determined by using phospho-PKA motif antibodies (Phospho-PKA substrates). E, Mek5^+/+ (Control) and Mek5^−/− fibroblasts were challenged with β-adrenergic receptor agonist isoproterenol (10 μm) for the times indicated. Activation of PKA was determined by using phospho-PKA motif antibodies (Phospho-PKA substrates). F, WAT isolated from adipo-ERK5^−/− and control mice were challenged with isoproterenol (1 μm) for 5 min. Levels of cAMP and protein concentration were determined by ELISA and colorimetric assays, respectively. Relative levels of cAMP production are shown (G). Tissue extracts from epididymal fat pads of 8-week-old adipo-ERK5^−/− and control mice were examined to determine the levels of phosphodiesterase activity. *, p < 0.05. Error bars, S.E.

FIGURE 9.

Opposing actions of ERK5 and calcineurin on NFATc4 and PDE4D. Conserved gate-keeping Ser residues are dephosphorylated by calcineurin phosphatase to promote nuclear localization of NFATc4. Nuclear NFATc4 then modulates gene transcription. Rephosphorylation of the conserved gate-keeping Ser residues of nuclear NFATc4 is carried out by ERK5. The opposing actions of ERK5 and calcineurin, therefore, regulate nucleo-cytoplasmic shuttling and subsequent NFATc4-mediated gene transcription. For PDE4D, dephosphorylation mediated by calcineurin stabilizes protein expression and increases cAMP hydrolysis. Hence, phospho-dependent degradation of PDE4D, which is triggered by GSK3β/CK1α phosphorylation, is attenuated upon calcineurin activation. Hydrolyase activity of PDE4D, however, is inhibited by ERK5 phosphorylation and thus decreases cAMP hydrolysis. Therefore, cAMP-mediated responses are opposed by calcineurin and ERK5 by regulating protein stability and enzyme activity of PDE4D, respectively.