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. 2015 Dec 1;22(6):997-1008.
doi: 10.1016/j.cmet.2015.09.029. Epub 2015 Nov 8.

Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure

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Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure

Kosaku Shinoda et al. Cell Metab. .
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Abstract

Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under β3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the β3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.

Figures

Figure 1
Figure 1. Identification of CK2 as a White Adipocyte-Selective Kinase Activated in Response to Norepinephrine and to a High-Fat Diet
(A) Schematic of phosphoproteomic analysis in brown, beige, and white adipocytes. (B) Hierarchical clustering and heatmap of phosphoproteome in brown adipocytes, white adipocytes (F442A), and beige adipocytes (F442A expressing PRDM16). Each column represents sample, and each row represents phosphorylated protein. The color scale shows the numbers of phosphorylation sites per phosphoprotein in blue (low) to yellow (high) color scheme. (C) Hierarchical clustering and heatmap of phosphoproteome in primary brown adipocytes, inguinal white adipocytes, and beige adipocytes (inguinal adipocytes treated with rosiglitazone). The color scale shows the peak intensity of phosphopeptides in log2 scale. The values are average of three measurements per sample. (D) Phosphopeptide motif analysis of brown, beige, and white adipocytes. Brown bars represent p values by repeated-measures ANOVA to show the difference in phosphoproteome containing the indicated kinase motif between brown and white adipocytes. Beige bars represent the difference between beige and white adipocytes. (E) Temporal dynamics of CK2 activity in brown, beige, and white adipocytes in response to norepinephrine treatment. Inset shows the conserved CK2-motif peptide sequences found in the phosphoproteome. n = 4. (F) CK2 activity in the inguinal WAT of mice under a regular chow diet or a high-fat diet. n = 10. (G) CK2 activity in the inguinal WAT, epidydimal → WAT, and interscapular BAT of mice under a regular chow diet and a high-fat diet for 16 weeks. *p < 0.05, and **p < 0.01 by one-sided Student’s t test.
Figure 2
Figure 2. Depletion of CK2 Activates the cAMP-Induced Thermogenic Program in White Adipocytes
(A) Expression of Ck2α1, Ck2α2, and Ck2β in inguinal adipocytes transfected with siRNAs targeting the indicated subunits or non-targeting control (Ctrl). n = 3. (B) Expression of thermogenic and adipogenic genes in inguinal white adipocytes depleted with each CK2 subunit. The cells were treated with forskolin at 10 μM for 4 hr to activate thermogenesis. n = 3. (C) Ucp1 expression in inguinal white adipocytes transfected with Ck2α1-targeting or control siRNAs. Differentiated cells were treated with for-skolin at 1, 10, and 50 μM for 4 hr. n = 3. (D) Expression of brown/beige fat-selective genes in inguinal white adipocytes transfected with Ck2α1-targeting or control siRNAs. n = 3. (E) Western blotting for UCP1 in inguinal white adipocytes transfected with Ck2α1-targeting or control siRNAs. Differentiated cells were treated with forskolin at 10 μM for 8 hr. β-actin was used as a loading control. (F) Cellular oxygen consumption rate (OCR) in inguinal white adipocytes transfected with Ck2α1-targeting or control siRNAs. Differentiated cells were treated with forskolin at 10 μM. n = 8. *p < 0.05, **p < 0.01, ***p < 0.001 by one-sided Student’s t test.
Figure 3
Figure 3. Pharmacological Inhibition of CK2 Promotes Beige Adipocyte Biogenesis In Vivo
(A) CK2 activity in the inguinal WAT of mice treated with vehicle or CX-4945 at 50 mg kg−1 twice daily for 5 days. n = 5. (B) Expression of brown/beige fat-selective genes in the inguinal WAT of mice treated with vehicle or CX-4945 for 5 days. n = 5. (C) Western blotting for UCP1 in the interscapular BAT and inguinal WAT in (B). β-actin was used as a loading control. (D) H&E staining of the inguinal WAT of mice treated with vehicle or CX-4945. Bottom shows high-magnification images. Scale bar, 100 μm. (E) UCP1 immunostaining (red) of the inguinal WAT in (D). DAPI (white) was used to stain nuclei. Scale bar, 70 μm. (F) Dose-dependent increase in Ucp1 mRNA expression in inguinal white adipocytes treated with CX-4945 at 20 nM and 100 nM (top) and CK2-VIII at 0.1 and 1 μM (bottom). Differentiated adipocytes were treated with 10 μM forskolin for 4 hr. n = 3. (G) Expression of brown/beige adipocyte-selective genes in cultured inguinal white adipocytes treated with CX-4945 at 100 nM. n = 3. (H) Expression of brown/beige adipocyte-selective genes in cultured inguinal white adipocytes treated with CK2-VIII at 1 μM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 by one-sided Student’s t test.
Figure 4
Figure 4. CK2 Blockade Activates Thermogenesis in White Adipocytes through Reduced CK2-Mediated Phosphorylation of Class I HDACs
(A) CK2-dependent phosphoproteome in white adipocytes. The color scale shows z-scored peak area of phosphopeptides in blue (low) to white to red (high) scheme. Pie charts show overlaps in the phosphoproteome between CK2 inhibitor-treated white adipocytes and beige adipocytes (up) or between CK2 inhibitor-treated white adipocytes and classical brown adipocytes (bottom). (B) Schematic of in vitro CK2 substrate profiling by LC-MS/MS. (C) GO analysis of phosphoproteins identified by in vitro CK2 substrate profiling in white adipocytes. The area of each pie slice represents the number of proteins that belong to the enriched GO terms (p < 0.01). (D) Venn diagram of the overlapped phosphoproteins identified by in vivo assay in (A) and in vitro CK2 substrate profiling in (B). (E) Phosphorylation status of class I HDACs in white adipocytes treated with CK2-VIII for 4 days. CK2-mediated phosphorylation of HDAC1 or HDAC2 was assessed by immunoprecipitation of respective antibodies followed by western blotting for phosphorylated CK2-substrate motifs. Total HDAC1 and HDAC2 proteins (input) were shown in the bottom panels. The HDAC-immunoprecipitates and input were derived from the identical samples and loaded into the separate gels. (F) Quantification of phosphorylation in HDAC1 and HDAC2 at indicated sites in inguinal white adipocytes incubated with CK2-VIII in the presence or absence of forskolin at 10 μM. (G) Conservation of phosphorylation sites on HDAC1 and HDAC2 in human, mouse, rabbit, and chicken. (H) Ucp1 and Elovl3 mRNA expression in inguinal white adipocytes incubated with CK2-VIII in the presence or absence of Mocetinostat at 150 nM and Tubastatin at 150 nM for 4 days. Differentiated adipocytes were treated with 10 μM forskolin for 4 hr. n = 3. **p < 0.01, ***p < 0.001 by one-sided Student’s t test.
Figure 5
Figure 5. CK2 Blockade Stimulates UCP1-Dependent Thermogenesis and Ameliorates Diet-Induced Obesity and Insulin Resistance
(A) VO2 of mice treated with vehicle and CX-4945 at indicated temperature. n = 5. p values were determined by repeated-measures ANOVA. (B) VO2 of wild-type (left) and Ucp1 null mice (right) treated with vehicle and CX-4945 under 22°C. n = 5. (C) Body weight of mice after treated with vehicle or CX-4945 for 40 days under a high-fat diet. n = 6. (D) Body fat percentage (left) and lean mass (right) of mice in (C) as assessed by Echo-MRI. (E) Weight of adipose tissues of mice in (C). (F) Glucose tolerance test in mice in (C). AUC is shown in the right panel. (G) Insulin tolerance test in mice in (C). AUC is shown in the right panel. (H) Fasting plasma insulin concentration in obese mice treated with vehicle or CX-4945 for 40 days under a high-fat diet. n = 5. (I) H & E staining of liver from mice treated with vehicle or CX-4945 for 40 days under a high-fat diet. Scale bar, 100 μm. (J) Liver triglyceride contents in mice in (I). n = 5 *p < 0.05, **p < 0.01 by one-sided Student’s t test.
Figure 6
Figure 6. ASO-Based Depletion of CK2 Synergistically Promotes Beige Adipocyte Biogenesis under Mild Cold Exposure
(A) Expression of Ck2α1, Ck2α2, and Ck2β in the inguinal WAT of mice treated with control or Ck2-ASO. n = 9. (B) Western blotting for CK2α1 in the inguinal WAT in (A). β-actin was used as a loading control. (C) CK2 activity in the inguinal WAT of mice treated with control or Ck2-ASO. n = 5. (D) VO2 of mice injected with control or Ck2-ASOs. The mice were treated with saline or CL316,243 at 0.5 mg kg1 under thermoneutrality. n = 5. (E) Expression of brown/beige fat-selective marker genes in the inguinal WAT of mice treated with control or Ck2-ASO under a high-fat diet. n = 5. *p < 0.05, ***p < 0.001 by one-sided Student’s t test. (F) H&E staining of the inguinal WAT of mice in (E). Lower-magnification images are shown in insets. Scale bar, 50 μm.
Figure 7
Figure 7. ASO-Based CK2 Depletion Enhances Cold-Stimulated Metabolism and Ameliorates Diet-Induced Obesity and Insulin Resistance
(A) Changes in body weight of mice treated with control or Ck2-ASOs under a high-fat diet. Mice were housed under 22°C or 17°C. n = 9. (B) Food intake in mice treated with control or Ck2-ASOs in (A). Data are average of the first 2 weeks. (C) Body fat percentage and lean mass in (A). (D) GTT of mice treated with control or Ck2-ASOs in (A). (E) AUC of GTT in (D). (F) ITT of mice treated with control or Ck2-ASOs in (A). (G) Fasting plasma insulin concentration of mice in (A). (H) Liver triglyceride contents in (A). *p < 0.05, **p < 0.01, ***p < 0.001 versus control by one-sided Student’s t test.

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