Presented on the research work by Chin-Chuan Chen, Chen-Hsin Kuo, Yann-Lii Leu, Shu-Huei Wang published in the Journal of Pharmacological Research

1. N. Vineeth
210604016
Department of Pharmacology
M.Pharm (III Sem)
Corylin reduces obesity and insulin resistance
and
promotes adipose tissue browning through
SIRT-1 and β3-AR activation
Pharmacological Research
Received 18 July 2020; Received in revised form 15 October 2020; Accepted 23 October 2020
Available online 27 November 2020
Table of
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Article

2. Introduction
Obesity
Global
epidemic
crisis
Cardiovascular
Disease
Diabetes
Insulin
resistance
Metabolic
syndrome
Cancers
Excess
calorie
intake
Insufficient
energy
dissipation
• Controlling energy intake had no effects for preventing and treating obesity
1

3. Need of alternative approach…..!
 Brown adipose tissue (BAT) activation or beige adipocyte induction in
White adipose tissue (WAT)(browning)
Mammalian
sirtuin 1
(SIRT1)
NAD+ & PPARγ dependent protein deacetylase
Regulates energy homeostasis
Reduces Obesity
Promotes Fat mobilization
Enhances Mitochondrial metabolism
2

4. β3-Adrenergic receptor
Beta 3 activation stimulates lipolysis in WAT & thermogenesis in BAT
3
WAT
BAT
BEIGE

5. CORYLIN
• Attenuates IL-6 or LPS induced inflammatory responses
• Stimulates osteoblast proliferation
• Supresses hepatocellular carcinoma progression
• Ameliorates hyperlipidemia & Insulin resistance
• Increases the sensitivity of hepatocellular carcinoma cells to
chemotherapy and radiotherapy
• Improves atherosclerosis and restenosis
(Corylin was kindly provided by Dr. Y. L. Leu)
4

6. Materials and methods
 Animals Male C57BL/6 (7WEEKS OLD)
*All 7 groups are fed upto 9 weeks
(IACUC No:CGU15-150)
S.No Groups Diet Treatment
1 1 Chow Vehicle
2 2 Chow Corylin 50mg/kg/day in 250mg/ml
3 3 60% HFD Vehicle
4 4(preventive group) High Fat Diet (HFD) Corylin50mg/kg
5 5(preventive group) High Fat Diet (HFD) Corylin100 mg/kg
6 6 Diet Induced
Obesity(DIO)
Vehicle
7 7(Treatment group) Diet Induced Obesity
(DIO)
Corylin 50mg/kg
5

7. Materials and methods
 Cell culture and differentiation
3T3-L1 preadipocytes grown at 370C in Dulbecco’s modified Eagle’s
medium(DMEM)
Containing 10% calf serum and penicillin streptomycin at 370C in an incubator
containing 5% CO2
Cells between passages 3 and 10 were used for the subsequent experiments
until confluence
6

8. • preadipocytes were induced to differentiate in DMEM including
1. 10 % fetal bovine serum (FBS)
2. MDI (0.5 mM 3-isobutyl-1- methylxanthine, 1μM dexamethasone 1μg/ml
insulin) with or without Corylin or dimethyl sulfoxide (DMSO).
• After 2 days, the medium was replaced with DMEM containing 10 % FBS
and 1μg/ml insulin with or without Corylin or DMSO every 2 days until day 8.
The undifferentiated cells were maintained in DMEM containing 10 % CS.
7

9. Materials and methods
Histological analysis of adipose tissue
The BAT, IWAT, AWAT, and EWAT were fixed with 4 % paraformaldehyde.
Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E).
Gene Expression
Total RNA was extracted using TRIzol, and 2μg of RNA was processed for
DNA digestion and reverse transcription. Gene expression was analyzed by
quantitative real-time PCR (qPCR) using SYBR Green and normalized to
β-actin 8

10. Materials and methods
Intraperitoneal glucose and insulin tolerance tests
For the intraperitoneal glucose tolerance test (IPGTT)
Mice were fasted for 16 h and then injected with 2g kg− 1 glucose solution at
time points of 0, 30, 60, 90, and 120 min to measure the value of blood
glucose
For the intraperitoneal insulin tolerance test (IPITT)
Mice were fasted for 5 h and injected with 1.2U kg− 1 insulin and sampled at
time points of 0, 30, 60, 90 and 120 min to measure the value of blood
glucose
9

11. Materials and methods
Tissue collection, Serum preparation and Serum parameter test
• The serum glucose, TG, TC, LDL-C and HDL-C levels were measured
using specific reagent kits (Randox, Antrim, UK).
• The serum alanine aminotransferase (ALT) level was measured using
specific reagent kits (Fortress Diagnostics, Antrim, Northern Ireland)
• Leptin and insulin was measured by ELISA 10

12. Materials and methods
Oil red O staining
The 3T3L1 adipocytes were fixed in 4 % paraformaldehyde, stained with
ORO for 15 min, and washed with tap water
The images of the ORO staining were recorded with a microscope.
To quantify the ORO staining, the ORO was eluted by isopropanol, and
the absorbance at 510 nm was measured by a spectrophotometer.
11

13. Materials and methods
WAT lipolysis assay
Incubated at 37◦C in the presence of Krebs-RingerHEPES buffer (135 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 0.4 mM K2HPO4, 20 mM HEPES, 1 mM
CaCl2 and 0.1 % glucose) containing 2 % fatty acid-free BSA and treated with
or without DMSO, Corylin (5 or 10 μM) or norepinephrine (10 ng/ml) for 4 h
Supernatants were collected, and the amount of glycerol released in the
medium was measured by a glycerol reagent kit 12

14. Materials and methods
Western blot analysis
The treated cell lysates (20μg) were subjected to SDS–PAGE and then
transferred to PVDF membranes
which were incubated with the appropriate primary antibodies overnight at
4◦C. Primary antibodies used:
UCP1 (1:1000, ab23841, Abcam),
SIRT1 (1:1000, sc-15404, Santa Cruz Biotechnology),
β3-AR (1:1000, #40617, SAB), ATGL (1:1000, GTX112141, GeneTex),
β-actin (1:10000, GTX109639, GeneTex)
α-tubulin (1:10000, GTX53688, GeneTex).
13

15. • Then the membranes were incubated with the HRP conjugated anti-
rabbit secondary antibodies for 1 h at room temperature.
• Immunoreactivity was detected with enhanced chemiluminescence and
quantified using Gel-Pro software.
Western blot analysis contd….
14

16. Materials and methods
Immunohistochemistry
• After fixation in 4 % paraformaldehyde, the fixed tissues were incubated
with the anti-UCP1 antibody (1:100, ab23841, Abcam) overnight at 4 ◦C.
• Then, the tissues were incubated with the AP conjugated anti-rabbit
secondary antibody for 1 h at room temperature
• Immunoreactivity was detected with ImmPACT Vector Red before
counterstaining with hematoxylin and examined under light microscope.
15

17. Materials and methods
Metabolic rate analysis
• Oxygen consumption (VO2), carbon dioxide production (VCO2) and
energy expenditure (EE) were determined for lean or obese mice fed with
or without corylin or vehicle for 9 weeks
SIRT-1 activity assay
• SIRT-1 activity of the 3T3L1 adipocytes was assessed using commercially
available kits (abcam).
16

18. Materials and methods
Transmission electron microscopy (TEM)
• The EWAT and BAT were fixed in 2 % glutaraldehyde and 2 %
paraformaldehyde
• Postfixed with 1 % osmium tetroxide for 1 h, dehydrated with ethanol and
then embedded in Epon and processed for TEM
• The best fit of Corylin in the activating domain of SIRT-1 and β3-AR was
obtained using the auto Dock Vina.
In silico analysis
17

19. 3) Results
3.1) Corylin ameliorates high-fat diet(HFD)-induced obesity
(A) Whole-body images of mice in different groups.
18

20. (B–C) The change in total body weight
(D–E) food intake
19
3.1) Contd…

21. (F–G) The weight of subcutaneous and visceral fat in mice with different treatments.
20
3.1) Contd…

22. Corylin treatment improves obese adipocytes through lipolysis stimulation.
(A) Representative micrographs of H&E-stained WAT sections in different groups. Higher magnification
images are shown on the right lower side of the image. 21
3.1) Contd…

23. (B–D) Adipocyte size was measured using Image-Pro Plus software. *P < 0.05 compared to the chow diet-fed
group. †P < 0.05 for Corylin-treated mice compared to the HFD or DIO group.
(E) Glycerol release was measured in the supernatant of Corylin-or norepinephrine-treated WAT.
22
3.1) Contd…

24. Corylin enhances energy expenditure
(A–B) VO2,(C–D)VCO2
23
3.1) Contd…

25. (E–F) EE and (G–H) RQ levels were measured in an indirect calorimeter.
24
3.1) Contd…

26. 3.2) Corylin alters the serum insulin, leptin, glucose and ALT levels
and improves insulin sensitivity in obese mice
(A–D) IPITT in mice after fasting for 6 h. Histograms represent the areas under the glucose curve 25

27. 3.2) Contd…
(E–H) IPGTT in mice after fasting for 16 h. Histograms represent the areas under the glucose curve.
26

28. 3.2) Contd…
(I–J) Fasting blood insulin, HOMA-IR and leptin levels of mice in different groups.
27

29. *P < 0.05 compared to the control group,
† P < 0.05 for Corylin-treated mice compared to the HFD group
‡ P < 0.05 for Corylin-treated mice compared to the DIO group.
The glucose, TC, LDL-C, TG and ALT concentrations were significantly increased in
the HFD fed mice compared with those in the control groups
28

30. 3.3) Corylin induces beige adipocyte differentiation and brown adipocyte
activation
(A) Confluent 3T3-L1 preadipocytes were cultured with MDI in the absence or presence of Corylin (5 or
10μM) for 8 days, as indicated in the experimental schematic model. (B) Representative bright field and ORO
staining photographs of 3T3-L1 cells showing intracellular lipid accumulation. (C) Quantitative analysis of
ORO staining and glycerol content 29

31. 3.3) Contd…
(D–F) Expression of browning-, mitochondrial biogenesis-, β-oxidation-, lipolysis- and beige-related genes in
3T3-L1 preadipocytes.
(G) Mature 3T3-L1 adipocytes were cultured and differentiated with MDI for 8 days and then cultured in the
absence or presence of Corylin (5 or 10μM) for another 3 days, as indicated in the experimental schematic
model. (H) Representative bright field and ORO staining photographs of 3T3-L1 adipocytes showing intracellular
lipid accumulation. 30

32. 3.3) Contd…
(I)Quantitative analysis of ORO staining and glycerol content.
(J)Expression of browning- and lipolysis-related genes in 3T3-L1 preadipocytes.
31

33. 3.3) Contd…
Expression of browning-, mitochondrial biogenesis-, β-oxidation-, lipolysis-, beige- and WAT-related genes in EWAT.
32

34. 3.3) Contd…
Expression of browning-, mitochondrial biogenesis-, and β-oxidation-related genes in BAT. 33

35. 3.3) Contd…
Representative micrographs of UCP1 staining and TEM in EWAT and BAT from mice after different treatments.
34

36. Rectal temperature was measured at the indicated times in mice with different treatments at 4 ◦C.
35
3.3) Contd…

37. 3.4 Corylin induced browning and lipolysis via SIRT-1 and β 3-AR-
dependent pathways
(A) Model of Corylin binding to SIRT1 and β3-AR.
(B) SIRT1 activity of 3T3-L1 adipocytes.
(C) Expression levels of Sirt1 and Adrb3 in EWAT and BAT from mice with
different treatments.
36

38. 3.4) Contd….
(D) mRNA and western blot analysis showing the SIRT1 and β3-AR levels in 3T3-L1 preadipocytes or mature
3T3-L1 adipocytes after MDI (M) and Corylin (10μM; 10C) treatment, as indicated in the experimental
schematic model.
37

39. 3.4) Contd….
(A) Confluent 3T3-L1 preadipocytes were cultured with MDI in the absence or presence of Corylin (10μM) or
EX527 (40μM; EX) or L748,337 (10μM; L) for 8 days, as indicated in the experimental schematic model.
Expression levels of browning- and lipolysis related genes (B) and proteins (C) and UCP1
immunofluorescence staining (D) in 3T3-L1 preadipocytes. 38

40. 3.4) Contd….
(E) Mature 3T3-L1 adipocytes were cultured and differentiated with MDI for 8 days and then cultured in the
absence or presence of Corylin (5 or 10μM) or EX527 (40μM) or L748,337 (10μM) for another 3 days, as
indicated in the experimental schematic model. Expression levels of browning- and lipolysis related genes (F)
and proteins (G) and UCP1immunofluorescence staining (H) in mature 3T3-L1 adipocytes. 39

41. Evidence that Corylin induces the formation of beige adipocytes in mouse
EWAT and BAT activation by increasing the expression of genes specific to
brown adipocytes and stimulating fatty acid oxidation
3.Results contd…
Primarily mediated by SIRT1 and β3-AR.
These data demonstrate that, in addition to reducing body weight and lipid
accumulation and stimulating lipolysis, a novel browning role of Corylin in
EWAT exists,
40

42. 4.Discussion
Corylin may protect against obesity progression by inducing browning and
lipolysis in WAT and thermogenesis in BAT through SIRT1 and β3-AR
regulation.
Corylin reduced obesity, enhanced the metabolic rate and improved
insulin resistance.
Results showed that Corylin increases the mRNA expression of brown
adipocyte-selective genes, such as ucp1, prdm16, and pgc1α, in EWAT,
BAT and 3T3L1 adipocytes.
41

43. Expression levels of beige adipocyte-selective markers such as in the
Corylin-treated EWAT or 3T3L1 adipocytes were much higher than those in
the HFD, DIO or MDI-treated group.
Activation of adrenergic signaling via β3-AR increases lipolysis in WAT,
and the mobilization of free fatty acids by lipolysis is essentially required for
thermogenesis in BAT & Beige.
The mRNA expression of mitochondrial biogenesis-related genes and
β-oxidation-related genes was also markedly elevated by Corylin treatment
4.Discussion contd…
42

44. • We found that Corylin could not improve obesity in ob/ob mice
• Corylin could not improve glucose tolerance and insulin sensitivity
• one is that Corylin is probably a leptin sensitizer; and other, the
present studies showed that Corylin induced WAT browning
probably through activation of β3- AR in MDI-treated 3T3L1 cells the
activation of WAT browning by Corylin cannot lead to the browning
function in ob/ob mice
Limitations
43

45. Future aspects
• Develop formulation
• Conduct more studies in humans
• Needs more disease models
• Psoralea coryliforia is used in the traditional drugs
• It grows throughout the plains of India, especially in the semi-arid regions of
Rajasthan and Punjab, adjoining Uttar Pradesh.
• It is also found throughout India in Himalayas, Dehra Dun, Oudh,
Bundelkhand, Bengal, Bombay, some valley in Bihar, Deccan, and
Karnataka 44

46. References
• Chen, C. C., Kuo, C. H., Leu, Y. L., & Wang, S. H. (2021b). Corylin
reduces obesity and insulin resistance and promotes adipose tissue
browning through SIRT-1 and β3-AR activation. Pharmacological
Research, 164, 105291. https://doi.org/10.1016/j.phrs.2020.105291
45