Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

<p>Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene <i>atg7</i> (<i>Atg7cKO</i>) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from <i>Atg7cKO</i> mice. In addition, <i>Atg7cKO</i> mice crossed with <i>Apoe</i> (apolipoprotein E)-deficient mice (<i>apoeKO; Atg7cKO:apoeKO</i>) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, <i>Atg7cKO: apoeKO</i> mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in <i>Atg7cKO:apoeKO</i> mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.</p

Zinc transporter ZIP13 suppresses beige adipocyte biogenesis and energy expenditure by regulating C/EBP-β expression

<div><p>Given the relevance of beige adipocytes in adult humans, a better understanding of the molecular circuits involved in beige adipocyte biogenesis has provided new insight into human brown adipocyte biology. Genetic mutations in <i>SLC39A13/ZIP13</i>, a member of zinc transporter family, are known to reduce adipose tissue mass in humans; however, the underlying mechanisms remains unknown. Here, we demonstrate that the <i>Zip13</i>-deficient mouse shows enhanced beige adipocyte biogenesis and energy expenditure, and shows ameliorated diet-induced obesity and insulin resistance. Both gain- and loss-of-function studies showed that an accumulation of the CCAAT/enhancer binding protein-β (C/EBP-β) protein, which cooperates with dominant transcriptional co-regulator PR domain containing 16 (PRDM16) to determine brown/beige adipocyte lineage, is essential for the enhanced adipocyte browning caused by the loss of ZIP13. Furthermore, ZIP13-mediated zinc transport is a prerequisite for degrading the C/EBP-β protein to inhibit adipocyte browning. Thus, our data reveal an unexpected association between zinc homeostasis and beige adipocyte biogenesis, which may contribute significantly to the development of new therapies for obesity and metabolic syndrome.</p></div

Adipocyte browning is accelerated in white preadipocytes from <i>Zip13</i>-KO mice.

<p>(A) Oil Red O staining of preadipocytes from WT and <i>Zip13</i>-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 <i>Zip13</i>-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 <i>Zip13</i>-KO mice (n = 3). (F) Time course of protein expression in WT and <i>Zip13</i>-KO preadipocytes after differentiation. Nuclear fractions were analyzed by immunoblotting. RNA Pol II was included as a loading control. Error bars show SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

ZIP13-mediated zinc flux negatively regulates adipocyte browning.

<p>(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) <i>MT1A</i> 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 <i>Zip13</i>-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 <i>Zip13</i>-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. *<i>p</i><0.05, **<i>p</i> < 0.01.</p

Schematic model of the role of ZIP13 in adipocyte browning.

<p>Zinc transport mediated by ZIP13 inhibits C/EBP-β accumulation, thereby negatively regulating adipocyte browning (left). Conversely, C/EBP-β accumulates in the <i>Zip13</i>-deficient condition (right).</p

C/EBP-β overexpression accelerates adipocyte browning independently of adipogenesis.

<p>(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 <i>aP2</i> (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 <i>aP2</i> (n = 4). (F) Schematic of the time course used in (G-J) using WT (WT) and <i>Zip13</i>-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 <i>aP2</i> (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 <i>aP2</i> (n = 4). Error bars show SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Upregulation of inguinal fat tissue browning and O₂ consumption rate in <i>Zip13</i>-KO mice.

<p>(A) H & E staining of inguinal fat and brown fat tissue in 10-week-old WT and <i>Zip13</i>-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 <i>Zip13</i>-KO mice. Scale bars = 100 μm. (C) Expression of the indicated genes in the inguinal fat tissue of 10-week-old WT and <i>Zip13</i>-KO mice (n = 5–6). (D) Expression of the indicated genes in the brown fat tissue of 10-week-old WT and <i>Zip13</i>-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 <i>Zip13</i>-KO mice (n = 3). (F) Energy expenditure of 10-week-old WT and <i>Zip13</i>-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. *<i>p</i> < 0.05, **<i>p</i> < 0.01 (WT vs. <i>Zip13</i>-KO), ^##<i>p</i> < 0.01 (WT STD vs. WT HFD).</p

ZIP13 negatively regulates adipocyte browning by stabilizing C/EBP-β.

<p>(A) C3H10T1/2 cells transfected with an siRNA targeting <i>Zip13</i> (si-<i>Zip13</i>-#1) or a non-targeting control (si-Ctrl) were stained with Oil Red O after induction of adipocyte differentiation. (B) Left panel: <i>Zip13</i> 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-<i>Zip13</i>-#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 <i>Zip13</i>-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. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p