Korean J Physiol Pharmacol.  2019 Sep;23(5):335-344. 10.4196/kjpp.2019.23.5.335.

Effects of caloric restriction on the expression of lipocalin-2 and its receptor in the brown adipose tissue of high-fat diet-fed mice

• Park KA
• Jin Z
• An HS
• Lee JY
• Jeong EA
• Choi EB
• Kim KE
• Shin HJ
• Lee JE
• Roh GS

• KMID: 2455810
• DOI: http://doi.org/10.4196/kjpp.2019.23.5.335

Abstract

Obesity causes inflammation and impairs thermogenic functions in brown adipose tissue (BAT). The adipokine lipocalin 2 (LCN2) has been implicated in inflammation and obesity. Herein, we investigated the protective effects of caloric restriction (CR) on LCN2-mediated inflammation and oxidative stress in the BAT of high-fat diet (HFD)-fed mice. Mice were fed a HFD for 20 weeks and then either continued on the HFD or subjected to CR for the next 12 weeks. CR led to the browning of the white fat-like phenotype in HFD-fed mice. Increased expressions of LCN2 and its receptor in the BAT of HFD-fed mice were significantly attenuated by CR. Additionally, HFD+CR-fed mice had fewer neutrophils and macrophages expressing LCN2 and iron-positive cells than HFD-fed mice. Further, oxidative stress and mitochondrial fission induced by a HFD were also significantly attenuated by CR. Our findings indicate that the protective effects of CR on inflammation and oxidative stress in the BAT of obese mice may be associated with regulation of LCN2.

Keyword

Brown adipose tissue; Caloric restriction; Lipocalin 2; Obesity

MeSH Terms

Adipokines
Adipose Tissue, Brown*
Animals
Caloric Restriction*
Diet, High-Fat
Inflammation
Lipocalins
Macrophages
Mice*
Mice, Obese
Mitochondrial Dynamics
Neutrophils
Obesity
Oxidative Stress
Phenotype
Adipokines
Lipocalins

Figure

• 

  Fig. 1 Effects of caloric restriction (CR) on high-fat diet (HFD)-induced white adipocyte-like phenotype in brown adipose tissue (BAT). (A) Schematic representation of experimental protocol. Male C57/6 mice were fed HFD for 20 weeks and then continued on the HFD or subjected to CR (2 g/day of HFD) for 12 weeks. (B) Representative images (×200) of H&E staining (upper panel) and immunofluorescence for perilipin (lower panel) in BAT sections. Nuclei were counterstained with DAPI. Arrow indicates lipid droplet. (C) Western blot analysis of perilipin protein expression using α-tubulin as a reference protein. (D) Triglyceride concentration in BAT. Data are presented as mean ± standard error of the mean; *p < 0.05 vs. normal diet (ND)-fed mice; †p < 0.05 vs. HFD-fed mice. Scale bar = 25 µm.

• 

  Fig. 2 Effects of caloric restriction (CR) on lipocalin 2 (LCN2), 24p3R expression, and iron accumulation in the brown adipose tissue (BAT) of high-fat diet (HFD)-fed mice. (A, B) Western blot analysis of LCN2 and 24p3R protein expression using α-tubulin as a reference protein. (C) Representative images (×200) of immunofluorescence for LCN2 and neutrophil elastase (NE) in BAT sections. Nuclei were counterstained with DAPI. (D) Representative images (×200) of iron staining in BAT sections. (E) The number of iron-positive cells in BAT sections. Data are presented as mean ± standard error of the mean; *p < 0.05 vs. normal diet (ND)-fed mice; †p < 0.05 vs. HFD-fed mice. Scale bar = 50 µm.

• 

  Fig. 3 Effects of caloric restriction (CR) on inflammation and oxidative stress in the brown adipose tissue (BAT) of high-fat diet (HFD)-fed mice. (A) Representative images (×200) of immunofluorescence for F4/80 and lipocalin 2 (LCN2) in BAT sections. (B) Western blot analysis of nuclear factor-kappa B (NF-κB) and nuclear respiratory factor-2 (Nrf2) protein expression, using p84 as a reference protein. (C, D) Western blot analysis of heme oxygenase-1 (HO-1) and superoxide dismutase1 (SOD1) protein, using α-tubulin as a reference protein. (E) Western blot analysis of mitochondrial ferritin (FtMt) protein expression, using voltage-dependent anion selective channel 1 (VDAC1) as a reference protein. Data are presented as mean ± standard error of the mean; *p < 0.05 vs. normal diet (ND)-fed mice; †p < 0.05 vs. HFD-fed mice. Scale bar = 10 µm.

• 

  Fig. 4 Effects of caloric restriction (CR) on mitochondrial fission in the brown adipose tissue (BAT) of high-fat diet (HFD)-fed mice. (A) Western blot analysis of phosphorylated dynamin like protein1 (pDrp1) (s616) and dynamin like protein1 (Drp1) protein expression, using voltage-dependent anion selective channel 1 (VDAC1) as a reference. (B) Western blot analysis of cytosolic Drp1 protein levels using α-tubulin as a reference protein. (C) Representative images (×200) of immunofluorescence for pDrp1 and VDAC1 in BAT sections. Nuclei were counterstained with DAPI. Data are presented as mean ± standard error of the mean; *p < 0.05 vs. normal diet (ND)-fed mice; †p < 0.05 vs. HFD-fed mice. Scale bar = 50 µm.

• 

  Fig. 5 Effects of caloric restriction (CR) on uncoupling protein 1 (UCP1) expression in the brown adipose tissue (BAT) of high-fat diet (HFD)-fed mice. (A) Western blot analysis of UCP1 protein expression, using α-tubulin as a reference protein. (B) Representative images (×200) of immunofluorescence for UCP1 in BAT sections. Nuclei were counterstained with DAPI. (C) mRNA expression of BAT-selective genes from BAT samples using qRT-PCR. Data are presented as mean ± standard error of the mean; *p < 0.05 vs. normal diet (ND)-fed mice; †p < 0.05 vs. HFD-fed mice. Scale bar = 10 µm.

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