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

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 mitochondria- Brown. These mitochondria have UCP-1 in their inner mitochondrial membrane.

Over view of BAT Thermogenesis

“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). 

 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). 3 groups – Vehicle, 50mg/kg curcumin or 100mg/kg curcumin. 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 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.

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.

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

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.

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

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

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

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

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

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

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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-reducing- obesity/#axzz3mtwcBM9u

Thank you Questions ??????

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