Fig 1.
Upregulation of inguinal fat tissue browning and O2 consumption rate in Zip13-KO mice.
(A) H & E staining of inguinal fat and brown fat tissue in 10-week-old WT and Zip13-KO mice. Scale bars = 100 μm. (B) Immunohistochemical staining of the UCP1 in inguinal fat and brown fat tissue sections from 10-week-old WT and Zip13-KO mice. Scale bars = 100 μm. (C) Expression of the indicated genes in the inguinal fat tissue of 10-week-old WT and Zip13-KO mice (n = 5–6). (D) Expression of the indicated genes in the brown fat tissue of 10-week-old WT and Zip13-KO mice (n = 5–6). (E) Heat map of mRNA levels of brown fat-specific, white fat-specific, and common fat genes in the iWAT from 10-week-old WT and Zip13-KO mice (n = 3). (F) Energy expenditure of 10-week-old WT and Zip13-KO mice during the light (left) or dark cycle (right) (n = 4–6). (G) Body weights of mice from 5 to 14 weeks of age when fed a standard (STD) or high-fat diet (HFD) (n = 7–9). Error bars show SEM. *p < 0.05, **p < 0.01 (WT vs. Zip13-KO), ##p < 0.01 (WT STD vs. WT HFD).
Fig 2.
Adipocyte browning is accelerated in white preadipocytes from Zip13-KO mice.
(A) Oil Red O staining of preadipocytes from WT and Zip13-KO mice in pro-adipogenic conditions. (B) Expression levels of the indicated genes in differentiated adipocytes in the presence or absence of forskolin (n = 3). (C) Total and uncoupled (oligomycin-insensitive) respiration of differentiated adipocytes (n = 3). (D) Differentiation of white preadipocytes from WT and Zip13-KO mice expressing an empty vector (Ctrl) or ZIP13-HA (ZIP13); mRNA levels of the indicated genes were measured using qRT-PCR (n = 4). (E) Time course of mRNA expression in differentiated white preadipocytes from WT and Zip13-KO mice (n = 3). (F) Time course of protein expression in WT and Zip13-KO preadipocytes after differentiation. Nuclear fractions were analyzed by immunoblotting. RNA Pol II was included as a loading control. Error bars show SEM. *p < 0.05, **p < 0.01.
Fig 3.
C/EBP-β overexpression accelerates adipocyte browning independently of adipogenesis.
(A) Diagram showing the time course used in the following experiments (B-E) using WT white preadipocytes expressing a control vector (WT Ctrl) or HA-C/EBP-β (WT C/EBP-β). These cells were differentiated using a white adipogenic cocktail (WW) or a brown adipogenic cocktail (WB). (B) Expression of the indicated genes was measured by qRT-PCR (n = 4). (C) The mRNA levels of white adipocyte markers related to (B) were normalized to that of aP2 (n = 4). (D) Expression levels of the indicated genes were measured by qRT-PCR (n = 4). (E) The mRNA levels of brown adipocyte markers related to (D) were normalized to that of aP2 (n = 4). (F) Schematic of the time course used in (G-J) using WT (WT) and Zip13-KO (KO) white preadipocytes. (G) Expression of the indicated genes was measured using qRT-PCR (n = 4). (H) The mRNA levels for white adipocyte markers related to (G) were normalized to that of aP2 (n = 4). (I) Expression of the indicated genes was measured using qRT-PCR (n = 4). (J) The mRNA levels of brown adipocyte markers related to (I) were normalized to that of aP2 (n = 4). Error bars show SEM. *p < 0.05, **p < 0.01.
Fig 4.
ZIP13 negatively regulates adipocyte browning by stabilizing C/EBP-β.
(A) C3H10T1/2 cells transfected with an siRNA targeting Zip13 (si-Zip13-#1) or a non-targeting control (si-Ctrl) were stained with Oil Red O after induction of adipocyte differentiation. (B) Left panel: Zip13 expression after the 2.5 days of transfection; Right panel: Expression of the indicated genes was measured using qRT-PCR (n = 4). (C) Protein expression of C/EBP-β. Tubulin was used as a loading control. (D) Protein expression of C/EBP-β in the presence of CHX. C3H10T1/2 cells were transfected with the si-Zip13-#1 or si-Ctrl oligonucleotide; β-actin is shown as a loading control. (E) C/EBP-β protein levels were quantified by normalization to the protein level at 0 h. Each dot shows two independent experiment results and lines show the average of the experiments. (F) HA-C/EBP-β immunoprecipitation, followed by immunoblotting to detect ubiquitin. (G) Protein expression of C/EBP-β in WT and Zip13-KO preadipocytes expressing scramble control (sh-con) or shRNA targeting C/EBP-β (shβ-1, or shβ-2); β-actin is shown as a loading control. (H) Expression of the indicated genes, measured by qRT-PCR (n = 4). Error bars show SEM. *p < 0.05, **p < 0.01.
Fig 5.
ZIP13-mediated zinc flux negatively regulates adipocyte browning.
(A) Amino acid alignment of TMD IV and V among selected members of the mouse ZIP family. The His residues in TMD IV and V (red) are putative zinc-binding sites that are highly conserved among ZIP-family members. (B) Expression of WT ZIP13 and ZIP13 mutants (H229A and H254A) in C3H10T1/2 cells; β-actin is shown as a loading control. (C) MT1A gene expression in C3H10T1/2 cells expressing WT and mutant (H229A and H254A) ZIP13 (n = 4). We have showed the results that appeared to be statistically significant against the WT background. (D) Immunoprecipitation of HA- or Myc-tagged WT, H229A, or H254A ZIP13, followed by immunoblotting for HA- or Myc-tagged ZIP13 to detect the homophilic characteristics of the ZIP13 mutants H229A and H254A. (E) Subcellular localization of ZIP13-HA (WT, H229A, or H254A) expressed in Zip13-KO preadipocytes. Cells expressing HA-tagged WT, H229A, or H254A ZIP13 (left panels) were double-stained with the Golgi apparatus marker GM130 (middle panels); the merged images are shown on the right. Scale bars = 40 μm. (F) Expression levels of the indicated genes in Zip13-KO cells expressing Ctrl, WT ZIP13, or the H229A or H254A ZIP13 mutant (n = 4). (G) Expression of C/EBP-β protein 4 days after differentiation; β-actin is shown as a loading control. Error bars show SEM. *p<0.05, **p < 0.01.
Fig 6.
Schematic model of the role of ZIP13 in adipocyte browning.
Zinc transport mediated by ZIP13 inhibits C/EBP-β accumulation, thereby negatively regulating adipocyte browning (left). Conversely, C/EBP-β accumulates in the Zip13-deficient condition (right).