Figure 1.
The Lcn2 expression in adipose tissue depots and liver during metabolic stress.
(A) the mRNA expression of Lcn2 in brown adipose tissue (BAT), epididymal adipose tissue (Epi), inguinal adipose tissue (Ing), and liver in C57/BL6 mice at 12 weeks of age during 48 h fasting. (B) Lcn2 protein expression in BAT, Epi and Ing adipose tissue in C57/BL6 mice at 12 weeks of age during 24 h fasting. (C) the mRNA expression of Lcn2 in BAT, Epi and Ing adipose tissue, and liver in C57/BL6 mice at 12 weeks of age after exposed to 22°C or 4°C for 4 h. (D) Norepinephrine induces Lcn2 expression and secretion in 3T3-L1 adipocytes. 3T3-L1 cells on day 7 of differentiation were treated with or without 1 µM norepinephrine (NE) for 24 h. Conditioned media and cells were collected and subjected to immune-blotting with the antibody against Lcn2. The mRNA expression levels in fasted and cold-adapted mice were normalized to the levels in control mice and shown as fold changes. The results are presented as mean ± SE and represent two independent experiments (n = 4–6 in each experiment). * p<0.05, ** p<0.01; * Comparison between control and fasted or cold-exposed mice.
Figure 2.
Regulation of Lcn2 mRNA expression and secretion by cytokines in adipocytes.
(A) the mRNA expression of Lcn2 in 3T3-L1 adipocytes treated with TNFα, IL-1β, or IL-6 at the concentration of 1 nM for 16 h. The mRNA expression levels in cytokine-treated adipocytes were normalized to the levels in control adipocytes and shown as fold changes. The results are presented as mean ± SE and represent two independent experiments. ** p<0.01, *** p<0.001; * Comparison between control and treated cells. (B) Lcn2 secretion in 3T3-L1 adipocytes treated with TNFα, IL-1β, or IL-6 at the concentration of 1 nM for 24 h. Conditioned media were collected and subjected to immune-blotting with the antibody against Lcn2. The results represent two independent experiments. (C) Western-blotting of Lcn2 in 3T3-L1 adipocytes treated with cytokines under the non-reducing condition.
Figure 3.
Regulation of Lcn2 expression and secretion by insulin and glucose in 3T3-L1 adipocytes.
(A) Dose-dependent effect of insulin on Lcn2 expression and secretion in the presence of high or low concentration of glucose. On day 7 of differentiation, cells were either maintained in DMEM containing high concentration of glucose (4.5 g/L) or switched to DMEM containing low concentration of glucose (1 g/L). Insulin was added at different concentrations as indicated. After 24 h, conditioned media and cells were collected and subjected to western-blotting with the antibody against Lcn2. (B and C) the quantification of band density shown in A. The results were presented as mean ± SE. (D) Effect of 3-O-methyl-d-glucose (Methyl-O-G) on insulin-stimulated Lcn2 expression and secretion. On day 7 of differentiation, cells were changed to high-glucose DMEM containing 4.5 g/L glucose or DMEM containing 1 g/L glucose and 3.5 g/L 3-O-methyl-d-glucose. Insulin was added at different concentrations as indicated. After 24 h, conditioned media and cells were collected and subjected to western-blotting. (E and F) the quantification of band density shown in D. The results were presented as mean ± SE.*or # p<0.05, ** p<0.01. *Comparison between control and insulin treatments. # Comparison between low and high glucose treatment or between Methyl-O-G and high glucose treatment.
Figure 4.
Effect of blocking NFκB signaling pathway activation on insulin and glucose induction of Lcn2 expression and secretion in adipocytes.
(A) 3T3-L1 adipocytes on day 7 of differentiation were treated with 5 mM aspirin in high-glucose DMEM with 100 nM insulin for 24 h. Conditioned media and cells were collected and subjected to western-blotting with the antibody against Lcn2. The quantification of band density shown in B and C. The results were presented as mean ± SE.*or # p<0.05, ** p<0.01. *Comparison between control and insulin treatment. # Comparison between control and Aspirin treatment. (D–F) 3T3-L1 adipocytes on day 7 of differentiation were pre-treated with various concentrations of BAY 11-7082 for 1 h followed by the treatment of 100 nM insulin for 24 h in the presence or absence of LPS. Conditioned media and cells were collected and subjected to western-blotting with the antibody against indicated proteins.
Figure 5.
Effect of fatty acids on Lcn2 expression and secretion in adipocytes.
3T3-L1 adipocytes on day 7 of differentiation were treated with 100 µM BSA with or without (A) 250 µM palmitate, 400 µM oleate, (B) 400 µM EPA, or (C) 10 µM phytanic acid in low-glucose media in the presence of insulin. After 24 h, conditional media and cells were collected and subjected to western-blotting with the antibody against Lcn2. The results represent for two independent experiments.