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1. 2016年月日
Phosphoproteomics Identifies CK2 as a Negative Regulator of
Beige Adipocyte Thermogenesis and Energy Expenditure
Cell MetabolismArticle
Kosaku Shinoda et al.
UCSF Diabetes Center and Department of Cell and Tissue Biology
IF:17.565, dx.doi.org/10.1016/j.cmet.2015.09.029
Xingyun Wang

2. Contents
1 Background
2 Results
3 Conclusions
4 Indications

3. Post-translational modification
Post-translational modifications are involved in almost all cellular events including:
Gene expression
Signal transduction
Protein-protein interactions
Cellular metabolism
Protein turnover and localization
DNA repair
Cell-cell interactions
Communication between internal and external cellular environment
Translocation of proteins across biological membrane
Background

4. Background
20-30% of all eukaryotic proteins can be
phosphorylated by over 500 kinases,
bringing the estimated number of phosphorylation sites to greater than 170,000.

5. Acetylation targets Lys residues in the amino-terminal tails of core histone proteins.
Background

6. Background

7. 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.
Background

8. Phosphoproteomics角度探究相同刺激（Catecholamines）对不同细胞（White/Beige/Brown）不同反应性的机制
Adipose-selective phosphorylation profiles
Brown adipocytes：NE signals through β-adrenoreceptors to increase
the expression and activity of Pgc-1α, a transcriptional coactivator that
coordinates gene programming in response to activation.
β-adrenergic stimulation triggers predominantly de novo differentiation
of precursor cells.
White and beige adipocytes:, caloric excess causes bipotent progenitors
to differentiate into white adipocytes, whereas β-adrenergic activators
stimulate beige adipocyte development.
Background

9. Results
Phosphoproteomic Profiling of Brown, Beige, and White Adipocytes
Phosphoproteomic Analysis in brown, beige, and white adipocytes.
Clustering and Heatmap of Phosphoproteome
总磷酸化水平，White组高于Brown、Beige组

10. Results
Phosphopeptide motif analysis
Phosphoproteomic Profiling of Brown, Beige and White Adipocytes
Relative activities of the predicted kinases were quantified by
counting the consensus phosphopeptide motifs at each time point.

11. Results
CK2 Pathway Is Preferentially Activated in White Adipocytes in
Response to Norepinephrine

12. Results
CK2 Pathway Is Preferentially Activated in White Adipocytes in Response to an Obesogenic Diet

13. Results
Depletion of CK2 Activates the cAMP-Induced Thermogenic Program in White Adipocytes
CK2 is a serine/threonine kinase that is composed of
catalytic subunits (α1 and α2) and two regulatory β
subunits

14. Results

15. Results

16. Results

17. Results
To ask if the CK2 inhibitor acts in a cell-autonomous fashion,inguinal white
preadipocytes were differentiated in the presence of two distinct classes of CK2
inhibitor, CX-4945 and CK2-VIII.
CK2-VIII: a highly selective CK2 inhibitor.
CX-4945: ATP-competitive inhibitor for CK2a1 and CK2a2 catalytic
subunits
Blockade of CK2 promotes the thermogenic program in WAT in vivo.

18. Results
Pharmacological Inhibition of CK2 Promotes Beige Adipocyte Biogenesis In Vivo

19. Results
Interscapular BAT and inguinal WAT
H&E staining

20. Results
inguinal WAT
UCP1 immunostaining (red)
DAPI (white)
These results further support the notion that CK2 is a major regulator of the white adipocyte-selective phosphoprotein profile

21. Results
Differentiated adipocytes were treated with
10 mM forskolin for 4 hr.

22. Results
CK2 Blockade Promotes Thermogenesis in White Adipocytes
through Reduced CK2-Mediated Phosphorylation of Class I HDACs

23. Results
CK2 is a major regulator of the white adipocyte-selective phosphoprotein profile

24. Results
Schematic of in vitro CK2
substrate profiling by LC-
MS/MS.
GO analysis of phosphoproteins
identified by in vitro CK2 substrate
profiling in white adipocytes
Venn diagram of the overlapped
phosphoproteins identified by in
vivo assay in and in vitro CK2
substrate profiling.

25. Results
Phosphorylation of class I HDACs (HDAC1 and HDAC2) was reduced by the CK2 inhibitor
Specifically, three phosphorylated sites in HDAC1 at serines 393, 421, and
423 and two sites in HDAC2 at serines 422 and 424 exhibited significant
changes in a CK2-dependent fashion
histone deacetylase

26. Results
Since inhibition of class I HDACs is known to increase
PGC1a expression and oxidative metabolism (Galmozzi
et al., 2013), we hypothesized that class I HDACs
mediate the CK2-linked adipocyte thermogenesis.

27. Results
Tubastatin, a selective inhibitor of HDAC6，that is
one of the class II HDACs but not a CK2 substrate
in adipocytes.
White adipocytes were incubated
with CK2-VIII in the presence or
absence of a class I HDAC-
selective inhibitor, Mocetinostat.
Class I HDAC-selective inhibitor, Mocetinostat.

28. Results
Pharmacological Blockade of CK2 Stimulates
UCP1-Dependent Thermogenesis and Ameliorates
Diet-Induced Obesity and Insulin Resistance

29. Results
Whole-body oxygen consumption rate (VO2)
The increase in VO2 level by CX-4945 was completely blunted in Ucp1 null mice , suggesting that a large part of the
increased energy expenditure by CK2 inhibition depends on UCP1-mediated thermogenesis, rather than heat loss.
C57BL/6J male

30. Results
Echo-MRI analysis showed that CX-4945
treatment significantly reduced body fat
mass without affecting lean body mass
Long-term treatment with CX-4945 was
effective to prevent diet-induced body
weight gain.
CX-4945 treatment significantly
reduced adipose mass of the
epididymal and inguinal WAT
depots

31. Results
Glucose tolerance test (GTT) found that mice treated
with CX-4945 exhibited moderately but significantly
lower blood glucose levels than vehicle-treated mice
Insulin tolerance test (ITT) showed that
CX-4945 treated mice displayed
significantly improved response to insulin
Serum insulin levels
under a fasted state

32. Results
Hepatic lower amounts of lipid droplets and triglyceride

33. Results
Pharmacological CK2 inhibition ameliorates dietinduced obesity and
insulin resistance by promoting UCP1-dependent thermogenesis.

34. Results
Antisense Oligonucleotide-Based Depletion of CK2 Synergistically Promotes Beige Adipocyte Biogenesis under Mild Cold
Exposure
Preated with ASO targeting Ck2a1 (Ck2-ASO) or control ASO at 40 mg kg, twice a week

35. Results
Ck2a1 was depleted in wild-type mice under a high-fat diet either at ambient
temperature or mild cold temperature . Consistent with the previous results,
Ucp1 mRNA in inguinal WAT was elevated by 3-fold and 5-fold by Ck2-ASO
treatment and mild cold exposure,respectively.
Whole body energy expenditure

36. Results
ASO-mediated CK2 depletion promotes
cold-induced thermogenesis in WAT.
Histological analysis of the inguinal WAT depots found that Ck2-
ASO and mild cold exposure synergistically promoted the
recruitment of multilocular and UCP1-positive beige adipocytes

37. Results
ASO-Based CK2 Depletion Enhances Cold-Stimulated Energy Metabolism and Ameliorates Diet-Induced Obesity and Insulin
Resistance
Ck2-ASO treatment at led to a 23% reduction of
dietinduced body weight gain as compared to control
mice at 22℃, whereas CK2-ASO or mild cold
exposure alone reducedbody weight gain by 13% and
12%, respectively.
No significant change in
food intake was detected
between Control and Ck2-
ASO treated group
The changes in body weight was due
to reduction in body fat mass, but not
to changes in lean mass

38. Results
While glucose tolerance was significantly improved in mice treated with Ck2-ASO, such improvement was further
enhanced under mild cold exposure.
GTT

39. Insulin sensitivity was significantlyimproved in mice treated with Ck2-ASO both under 22℃ and 17℃
ITT
Results

40. Conclusions
1.CK2 is preferentially activated in white fat by norepinephrine
and under obesity.
2.CK2 inhibition in white fat activates thermogenesis in
response to cAMP stimuli.
3.CK2 inhibition increases UCP1-dependent energy.
4.CK2 inhibition ameliorates diet-induced obesity and insulin
resistance.

41. Indications
1.好坏脂肪的转化：
药物刺激白色脂肪棕色化为新近研究热点和方向——白色脂肪，储存能量并与糖尿病和肥胖症有关；棕色脂肪，通过
燃烧能量产生热量。
寒冷刺激、运动等因素可促进棕色化的过程，临床上尚无促进转化的药物。
CK-2被誉为棕色脂肪开关
冬眠的动物（如熊）即使不运动也积累了大量的棕色脂肪。
“睡觉也能棕色化？”
2.总体水平的磷酸化、去磷酸化到组蛋白去乙酰化酶位点的确定：
针对于我们的研究：蛋白酶体抑制剂可以抑制整体水平的蛋白/多肽的降解；进一步研究的方向。

42. 谢 谢