Direct and Indirect effects of Guggulsterone on the Induction of Beiging in Mature 3T3-L1A Adipocytes B 1 1 5 t Control GS 6 µM GS 25 µM 1 2 2 2 2 n 2 e Colette N. Miller, Yusra Azhar, Ashish Parmar, Janaiya Samuels, Rangaiaht 1 1 0 Shashidharamurthy,* Srujana Rayalam n PGC1a ) l A o c o r 1 2 l t 1 0 5 Department of Foods and Nutrition, University of Georgia, Athens, GA Department of Pharmaceutical Sciences, School of a Pharmacy, Philadelphia College of Osteopathic Medicine- GA Campus, Suwanee, GA i Cn o n tro l IS O G S 6 u M G S 2 5 u M PPARy r o d c n 1 0 0 o % ( h U C P 1 c B-Actin o t 9 5 i Abstract Direct Effects of GS on 3T3-L1 AdipocytesM GS signaling 9 0 T B X 1 Phytochemicals have long demonstrated anti-obesity properties in C o n tro l G S 2 5 u M Phytochemicals may induce beiging through nuclear receptor activation. adipocytes. Their ability, however, to induce browning in white adipose GS induces mitochondrial biogenesisC D T u b lin tissue is only beginning to emerge. We have recently established that the white adipocyte cell line, 3T3-L1, is capable of beiging under beta- 5 0 0 2 0 0 Control Iso 10mM p = 0 .1 8 adrenergic conditions. Using this information, we sought to investigate if the B p = 0 .1 8 A 4 0 0 1 5 0 ) plant steroid guggulsterone (GS) can induce beiging in 3T3-L1s. 3T3-L1 ) l l o o r preadipocytes were differentiated using established protocols r C o n tro l IS O G S 6 u M G S 2 5 u M t t 3 0 0 1 R n supplemented with rosiglitazone and thyroid hormone. Direct effects of GS n C A o o 1 0 0 P c c G were measured by treating mature 3T3-L1s for 24 hours. Indirect effects 2 0 0 P U C P 1 P % % ( ( were measured by treating mature 3T3-L1s with conditioned media from GS 6.25μM GS 25μM 5 0 GS-treated RAW 264.7 macrophages. Direct treatment of 3T3-L1s with GS 1 0 0 resulted in increased lipolysis, increased mitochondrial activity (11%), and T B X 1 increased peroxisome proliferator-activated receptor gamma coactivator 1- 0 0 C o n tro l G S 6 u M G S 2 5 u M C o n tro l G S 6 u M G S 2 5 u M alpha (PGC1α) levels by 250% more than control. 3T3-L1s also T u b liAn demonstrated an increase in uncoupling protein 1 (UCP1) expression by GS induces adipocyte beiging 200% and beige marker, T-box protein 1 (TBX1) expression by 80% more B C than control. Furthermore, this was accompanied by increased levels of G 4 0 0 GS treatment appears to protein-coupled bile acid receptor (TGR5) and its downstream target * A upregulate TGR5-DIO2 pathway iodothyronine deiodinase 2 (DIO2). Treatment of RAW 264.7 macrophages * C o n tro l IS O G S 6 u M G S 2 5 u M in 3T3-L1s. 3 0 0 ) ) with GS induced a 60% increase in catecholamine release into the media ) l l l o o o r r compared to control. Using this conditioned media from macrophages, 3T3- U C P 1 r 1 1 1 t t t P P P n n L1 adipocytes increased the expression of DIO2 and UCP1 following 24 n 2 0 0 o o o C C C c c c U U U hours of incubation. Results from this study demonstrate that GS can T B X 1 % % % ( ( potentially induce beiging in white adipose tissue through two distinct ( 1 0 0 mechanisms: (1) direct signaling through the TGR5-cAMP-DIO2 pathway T u b lin B and (2) indirectly through stimulating catecholamine release in Working Model macrophages. Thus, it is reasonable to conclude that GS may improve the B 0 A C o n tro l IS O G S 6 u M G S 2 5 u M metabolic capacity of adipose tissue thereby counteracting the effects of C 1 2 0 1 0 0 obesity. * * * ) ) y y l 3 0l 0 t t * * * * i i 8 0 o o l l i i r r t t * b b n n a a i i 6 0 o o v v c c l l l l e e % % 4 0 ( ( C C ) ) ) l l l 2 0 0 * o o o 2 0 r r r 1 1 Indirect Effects1 of GS on 3T3-L1 Adipocytes t t Research Goals t n n n X X X 0 o o o B B B l o M M M M M c c c r u u u u u T T • Determine if the anti-obesity phytochemical, guggulsterone, can T t n 1 3 6 .5 5 o 2 2 C S S S G G G 1 S % % A GS protects against% 1 0 0 LPS-injury in RAWG 264.7 macrophages S ( ( promote mitochondrial biogenesis and beiging in 3T3-L1 adipocytes. ( G C C • Investigate if guggulsterone1 2 0 mediates mitochondrial biogenesis through direct or indirect signaling. B 1 0 0 102 0 * * * * * * * ) y 1 0 0 C o n tro l IS O G S 6 u M G S 2 5 u M# l t i 8 0 o l ) ) y y i l l r t t i i t 8 0 o o l l b i i r r n t t a b b i * * * * n n * * * * a 6 0 a o i i v 6 0 o o v v c c c l l l l l l e e e % % 4 0 % ( ( C Direct4 0 Effects of GS on C ( C 2 0 2 0 0 l 3T3-L1 Adipocytes o L M M M M r 0 t /m u u u u n g 1 6 1 6 o u S S S S C o n tro l G S 6 u M G S 1 2 .5 u M G S 2 5 u M C 1 G G G G Conclusions S + + P L S S P P L L B GS appears to have 2 distinct effects on adipocyte beiging: GS decreases adipogenesis C GS induces catecholamine secretion in RAW 264.7 macrophages that can upregulate 1) Direct signaling in adipocytes leading to upregulation of thermogenic/ 2 0 0 5 0 0 n 1 2 0 n UCP1 in adipocytes beiging makers and enhanced mitochondrial biogenesis. This effect o o i i e e t t 4 0 0 * * * * c c s s appears to be partly mediated through TGR5 signaling. u * * * * u ) ) a a l 1 0 0 l d d o o e e o o r r t l l r r 3 0 0 t 1 5 0 t 2) Indirect signaling in adipocytes through macrophage M2 ) p p ) ) e e n n n l l l r r o o e e e 8 0 # o t o o c c d d i i r e e 2 0 0 polarization and catecholamine secretion. r r t n x x t t % % n n o o ( ( o n i i * * * * n n c c c 6 0 o i 1 0 0 i o o 1 0 0 r r m m * * * * t t c * * * i i d c c a a i l l N N p % o 4 0 o i 0 ( % % h h ( ( L l o L M M M c c r * * * * 5 0 t /m u u u e e n g 6 5 5 o 2 2 t 2 0 t u C S 1 G S S a a S G G Financial Support P + C C *p<0.05 L S 0 P 0 L **p<0.01 C o n tro l G S 6 u M G S 1 2 .5 u M G S 2 5 u M ***p<0.001 Funding was provided by the Philadelphia College of Osteopathic Medicine C o n tro l IL 4 G S 6 u M ****p<0.0001 Biomedical Research Program. #p<0.05 vs. + control.