1. Biochemical and Biophysical Research Communications (2015)
Curcumin promotes browning of white adipose tissue in a norepinephrine-dependent way
Biochemical and Biophysical Research Communications (2015)
Keerthi Gullapalli, Srilatha Sakamuru and Nooshin Safari
BINF 704 Fall 2015

2. Over view Introduction Methods Results Discussion
Future Implications of Curcumin in Obesity

3. INTRODUCTION
Adipose tissue- loose connective tissue composed of adipocytes.
2 types – Brown adipose tissue (BAT)
White adipose tissue (WAT)
White adipose tissue (WAT):
20-25% of the body weight in humans.
Store of energy.
Single large fat droplet, with nucleus at the rim of the cell.

4. Brown adipose tissue (BAT):
Abundant in newborns and hibernating animals.
In neonates, brown fat makes up about 5% of body mass.
In adults – upper chest and neck- supraclavicular region.
Generates body heat during cold exposure by thermogenesis.
Contain small droplets with large number of mitochondria- Brown.
These mitochondria have UCP-1 in their inner mitochondrial membrane.

7. Over view of BAT Thermogenesis

10. “Brown like adipocytes”- Beige Adipocytes
Clusters of UCP-1 adipocytes with thermogenic capacity also develop in white adipose tissue (WAT) in response to various stimuli.
These adipocytes are called ‘beige’ or ‘brown like adipocytes’.
Brown adipocytes are located in dedicated depots and express constitutively high levels of thermogenic genes.
Inducible brown like adipocytes – Beige cells, develop in white fat in response to various activators.
Brown adipocytes express high levels of Ucp1 and other thermogenic genes under basal (unstimulated) conditions.
Beige adipocytes express these genes only in response to activators such as agonists of the β-adrenergic receptor or peroxisome proliferator- activated receptor-γ (Ppar-γ). 

11.  Similar to adipocytes of BAT, beige cells also have multi-locular lipid droplet morphology, high mitochondrial content and expression of core set of brown fat specific genes such as ucp-1 and have the ability to burn fat.
Number of beige cells is inversely correlated with body mass index (BMI) in humans.
Therefore, identification of factors that can induce the browning of white fat represents an attractive potential strategy for the management and treatment of metabolic diseases, including obesity and Type 2 diabetes.

12. Curcumin (Diferuloylmethane)
Curcumin is a natural flavonoid compound in turmeric.
Safe and tolerable at high doses of 12g/day in humans.
When pharmacologically administered to insulin-resistant obese rodents, curcumin increased weight loss, improved insulin sensitivity and normalized carbohydrate and lipid parameters.
In pre-diabetic population, curcumin prevented type-2 DM development and also improved overall functioning of β-cells.
Anti-Obesity and Anti-Diabetic properties.

13. Methods Animal experiments: Male C57BL/6 mice (27 ±2 g).
22 ± 2°c, 55± 5 % relative humidity, 12-h light/dark cycle. (Food and water were provided).
3 groups – Vehicle, 50mg/kg curcumin or 100mg/kg curcumin.
Curcumin dissolved in corn oil ( mg/kg body weight) was administered to the mice for 50 consecutive days.
Corn oil was administered to vehicle group mice.
Body weight recorded every 5 days and food intake was measured every other day through out the study.

14. Cold exposure:
Animals placed in cold for 6h at 4°c without access to food and water and temp was recorded daily with a rectal probe connected to a digital thermometer.
Histology and immunochemistry:
Inguinal and epididymal adipose tissues were harvested and fixed in 4% formaldehyde for 24hrs.
Tissues were washed with PBS, stored in 70% ethanol and embedded in paraffin.
Sections subjected to H & E staining and immunostaining with UCP-1 antibody.

15. mtDNA quantification:
Total DNA was extracted from inguinal adipose tissues using DNeasy blood and tissue kit
Results were calculated as difference in threshold cycle (ΔCT) values from mtDNA compared with nuclear DNA by qPCR.
Data are expressed as 16srRNA normalized to hexokinase-2 gene.

16. Western blot analysis:
Tissues were harvested and lysed in RIPA buffer containing a protease inhibitor.
Total protein lysates subjected to SDS- PAGE gels and transferred to PVDF membranes.
Membranes were incubated with appropriate primary antibodies against UCP-1 or PGC1-α.
Secondary antibodies were detected using infrared imaging system.

17. Q-RT PCR analysis:
Total RNA was extracted from the tissue using TRIZOL method.
Reverse transcribed to cDNA and qRT PCR was performed with SYBR Green PCR MasterMix.
The relative amount of mRNA normalized to b-actin was calculated using delta- delta method.

18. Plasma nor epinephrine measurement:
Blood sample from each mouse centrifuged at 3000 rpm for 10 min.
The extracted plasma was frozen at -80c.
Plasma norepinephrine level was measured using mouse norepinephrine ELISA kit.

19. Results Assessment of weight gain and fat mass in mice treated with Curcumin
Mice treated with 50 or 100 mg/kg curcumin
were noticeably protected from weight gain
Mice treated with 50 or 100 mg/kg curcumin had less fat mass than control mice

20. Results Assessment of food intake and body temperature during cold exposure in mice treated with curcumin
Mice treated with 50 or 100 mg/kg curcumin
did not alter food intake
Treated mice exhibited increased body temperature compared with control mice during 6 h of exposure to cold.

21. Results Curcumin induces browning of inguinal WAT
Hematoxylin and eosin stained sections of inguinal WAT from mice treated with vehicle or curcumin.
UCP1 protein (brown stain) immunohistochemistry in inguinal WAT from mice treated with vehicle or curcumin

22. Results Western blot and Quantitative PCR analysis
Curcumin (50 or 100 mg/kg) induced the expression of a number of brown fat-specific genes in iWAT, including Ucp1, Pgc1a, Prdm16, Dio2, Ppara, Cidea, Elovl3, Nrf1, mtTfa, and ATPsyn,
Western blots demonstrating key protein changes in inguinal WAT after curcumin treatment
Curcumin (50 or 100 mg/kg) increased
mitochondrial biogenesis as determined by mtDNA copy number

23. Results Curcumin administration for 50 days does not induce brown-like adipocyte in epididymal adipose tissue
epididymal WAT (eWAT) from mice treated with curcumin (50 or 100 mg/kg) had decreased white adipocyte size compared to controls
But, morphological beige adipocyte was not seen in the eWAT after curcumin (50 mg/kg or 100 mg/kg) treatment

24. Results Curcumin administration for 50 days does not induce brown-like adipocyte in epididymal adipose tissue
qPCR analysis revealed that curcumin (50 or 100 mg/kg) did not induce the expression of a number of brown fat-specific genes, including Ucp1,
Pgc1a, Prdm16, Dio2, and Cidea
Western blot analysis showed that eWAT from curcumin-treated and control mice expressed similar levels of UCP1

25. Results Effect of 50 days curcumin treatment
β3AR gene expression in inguinal WAT
β3AR mRNA in inguinal WAT was measured by qRT-PCR
Plasma norepinephrine levels
Norepinephrine levels in plasma were
measured by ELISA

26. Discussion
A strategy to increase energy expenditure, stimulating the development of beige adipocytes in WAT represents an attractive concept for combating obesity and associated metabolic diseases.
Browning agents have been described and they increase UCP1 activity and consequently heat production, leading to slimming.
Intragastric administration of curcumin (browning agent) exhibited
lower body weight gain and less fat mass.
Moreover, UCP1-positive brown fat-like cells emerged in iWAT of mice after curcumin treatment.
Did not affect food intake in mice, suggests that curcumin alters energy metabolism.
Induces thermogenic gene expression, beige adipocyte emergence and mitochondria biogenesis in iWAT

27. Discussion
Although curcumin stimulates UCP1 gene expression and beige cells emerged in iWAT, but not in eWAT.
Demonstrated that curcumin increases β3AR gene expression in iWAT, so it induces WAT browning via the norepinephrine-β3AR pathway.
additional studies will be required to determine the source of the increased norepinephrine induced by curcumin.
Through this study, a clear function of curcumin observed was:
Regulating adaptive thermogenesis by increasing the expression of thermogenic genes and beige cells emerging in inguinal WAT.
This establishes an important role for curcumin in browning white fat.
So, curcumin can be proposed to be used for therapy in obese patients.
Its effects on body weight, lipid metabolism and β-cell function have evoked its use in treatment for diseases, such as diabetes.

28. Discussion
Turmeric has been long recognized for its anti-inflammatory and health- promoting properties.
Curcumin is one of the principal anti-inflammatory and healthful components of turmeric comprising 2-8% of most turmeric preparations.
Curcumin’s medicinal use dates back 6,000 years.
Ancient Egyptian pharaohs and Ayurvedic medical practitioners in India first discovered its beneficial properties.
Today, researchers from many fields are eager to study curcumin’s many effects
Various experiments on mouse models concluded that orally ingested curcumin Significantly Improves Obesity-Associated Inflammation and Diabetes.

29. Future implication of curcumin in obesity
Review Article
Curcumin and Obesity
Department of Pharmacology and Toxicity ,School of Medicine and Biochemical Sciences, State University of New York at Buffalo
Nooshin Safari
BINF 704 Fall 2015

30. Outlines: Introduction
Curcumin Effects on Obesity Associated Inflammation
Curcumin Effects on Adipocyte Differentiation
Curcumin Effects oβn Wnt/β- Catenin Signaling 
Curcumin effects on the NRF2/KEAP1 pathway
Curcumin and epigenetics
Conclusions

31. Introduction Curcumin is photochemical component in Turmeric
Potential therapeutic activity in the treatment of obesity and obesity -related metabolic disorder

32. General function of curcumin
Anti inflammatory effects
Induces expression of adiponectin
Reduces expression of the potent proinflammatory adipokines Tumor Necrosis factor- α (TNF α)
Chemo attraction effect on Monocyte protein 1
Inhibits plasminogen activator type1
Inhibits adipocyte differentiation
Promotes antioxidant activities

33. Curcumin effects on obesity associated inflammation
Curcumin has effects on adipose tissue in obesity

34. Down –regulates the DNA binding and transcriptional activities of inflammatory transcription factors NF-kB and AP-1
Scavenges reactive oxygen species
Suppresses mitogen-activated protein kinases
Inhibits inflammatory cytokine secretion
Inhibits LPS stimulation

35. Curcumin Effects on Adipocyte Differentiation
Suppresses preadipocyte differentiation
Reduces expression of adipocyte transcription factors(C/EBPα,PPARγ) and glycerol-3-phosphate acyl transferase-1 also synthesis of glycerol lipids
Increases the level of phospho-AMPK

36. Curcumin effects on Wnt/β-catenin signaling
Controls the fate of mesenchymal stem cells
Restores nuclear translocation of β-catenin in a dose dependent manner

37. Curcumin effects on the NRF2/KEAP1 pathway
Affects the NFE2-related factor 2(Nrf2)
Nuclear translocation of Nrf2 in hepatocytes
Activates Nrf2
Promotes antioxidant expression

38. Curcumin and Epigenetics
specific adverse environmental exposures which affect genome directly lead to higher level of susceptibility and consequent diseases such as:
Type two diabetes
Cardiovascular diseases
Promotes epigenetic modulation
DNA hypo methylation

39. Conclusion Suppress inflammation
Inhibits preadipocyte differentiations
Activates potent cellular antioxidant (in other tissues )
Improves hyperglycemia and insulin resistance
Inhibits macrophage infilteration

40. References
S. Wang, et al., Curcumin Promotes Browning of White Adipose Tissue in a Norepinephrine-Dependent Way. Biochemical and Biophysical Research Communications (2015),
Peter G. Bradford, Curcumin and Obesity. Biofactors (2013),
Weisberg SP, Leibel R and Tortoriello DV. Dietary Curcumin Significantly Improves Obesity-Associated Inflammation and Diabetes in Mouse Models of Diabesity. Endocrinology (2008),
obesity/#axzz3mtwcBM9u

41. Thank you
Questions ??????

42. General function of curcumin
Anti inflammatory effects
Inducing expression of adiponectin
Reducing expression of the potent proinflammatory adipokines Tumor Necrosis factor- α (TNF α)
Chemotaxis for Monocyte protein 1(PAI-1)
Inhibiting plasminogen activatortype1(PAI-1)
Inhibiting adipocyte differentiation
Promoting antioxidant activities