Download Srilatha Sakamuru, Keerthi Gullapalli

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
Over view
▪ Introduction
▪ Methods
▪ Results
▪ Discussion
▪ Future Implications of Curcumin in Obesity
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.
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 mitochondriaBrown.
▪ These mitochondria have UCP-1 in their inner mitochondrial
membrane.
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Over view of BAT Thermogenesis
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“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 proliferatoractivated receptor-γ (Ppar-γ).
▪ 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.
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.
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).
curcumin.
3 groups – Vehicle, 50mg/kg curcumin or 100mg/kg
Curcumin dissolved in corn oil (50- 100 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.
▪ 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.
▪ 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.
▪ Western blot analysis:
Tissues were harvested and lysed in
RIPA buffer containing a protease
inhibitor.
Total protein lysates subjected to SDSPAGE 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.
▪ 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 deltadelta method.
▪ 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.
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
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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.
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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
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Results
Western blot and Quantitative PCR analysis
Western blots demonstrating key protein
changes in inguinal WAT after curcumin
treatment
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,
Curcumin (50 or 100 mg/kg) increased
mitochondrial biogenesis as determined
by mtDNA copy number
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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
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Results
Curcumin administration for 50 days does not induce brown-like adipocyte in
epididymal adipose tissue
Western blot analysis showed that eWAT
from curcumin-treated and control mice
expressed similar levels of UCP1
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
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Results
Effect of 50 days curcumin treatment
Plasma norepinephrine levels
β3AR gene expression in inguinal WAT
Norepinephrine levels in plasma were
measured by ELISA
β3AR mRNA in inguinal WAT was measured by
qRT-PCR
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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
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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.
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Discussion
▪ Turmeric has been long recognized for its anti-inflammatory and healthpromoting 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.
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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
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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
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Introduction
▪ Curcumin is photochemical component in Turmeric
▪ Potential therapeutic activity in the treatment of obesity and
obesity -related metabolic disorder
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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
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Curcumin effects on obesity associated inflammation
Curcumin has effects on adipose tissue in
obesity
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▪ 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
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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
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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
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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
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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
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Conclusion
▪ Suppress inflammation
▪ Inhibits preadipocyte differentiations
▪ Activates potent cellular antioxidant (in other tissues )
▪ Improves hyperglycemia and insulin resistance
▪ Inhibits macrophage infilteration
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References
▪ S. Wang, et al., Curcumin Promotes Browning of White Adipose Tissue in a
Norepinephrine-Dependent Way. Biochemical and Biophysical Research
Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.018
▪ Peter G. Bradford, Curcumin and Obesity. Biofactors (2013),
http://www.ncbi.nlm.nih.gov/pubmed/23339049
▪ Weisberg SP, Leibel R and Tortoriello DV. Dietary Curcumin Significantly
Improves Obesity-Associated Inflammation and Diabetes in Mouse Models of
Diabesity. Endocrinology (2008),
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2453081/
▪ http://undergroundhealthreporter.com/curcumin-benefits-many-in-reducingobesity/#axzz3mtwcBM9u
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Thank you
Questions ??????
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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
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