A microrna-34a/fgf21 Regulatory Axis and Browning of White Fat

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A microrna-34a/fgf21 Regulatory Axis and Browning of White Fat Jongsook Kim Kemper, Ph.D Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, USA 213 International Conference on Diabetes and Metabolism and 5 th Asian Association for the Study of Diabetes, Seoul, Korea

The Global Epidemic of Obesity > 1/3 of adult population in USA are obese and 2/3 are overweight. Child obesity is rapidly growing.

Obesity: Disruption in Energy Balance Energy Intake Energy Balance Energy Expenditure Basal Metabolism Adaptive Thermogenesis Physical Activity Obesity and the regulation of energy balance, Review, by B. Spiegelman and J. Flier, Cell, 21

White Adipose Tissue (WAT) & Brown Adipose Tissue (BAT) Adipose tissue (fat cell) is an endocrine organ that control energy balance. WAT (Bad Fat): Stores excess energy as Fat (TG). In particular, abdominal Fat BAT (Good Fat) Packed w/ mitochondria Dissipates energy as heat (via UCP1). Newborns and small animals have BAT. Also, adults have BAT (cold exposure)! Activation of BAT would be an appealing option for weight-loss and fighting obesity. BUT!!!

Third Type of Fat: Beige Fat Browning of WAT P. Searle, U Penn

Third Type of Fat: Beige Fat Beige Fat: Stained by an antibody against UCP1, by Ting Fu HFD HFD (+anti-mir-34a) Beige fats are detected by brown-fat specific UCP1 staining. Beige fats are distinct from the classical BAT (gene expression patterns, basal levels UCP1, myogenic myf5-negative cell lineage, -----). Beige fat depots are developed in WAT in response to various activators, including transcriptional regulators, hormones, and cold exposure (PRDM16, irisin, FGF21, cold exposure (activators of β-adrenergic receptor)-----).

Fibroblast Growth Factor-21 (FGF21) Upon cold exposure, FGF21 promotes fat browning in PGC-1a dependent manner. Both systemically and locally acts on WAT/BAT and liver to beneficially control metabolism and energy balance. Binds to the adipocyte FGF21 membrane receptor complex, FGFR1 and βkl. FGF21 resistance in obesity was proposed, but the mechanisms are not clear. FGF21 FGFR1 βkl C. Cantor and J., Auwerx, review, Science, 212 Oxidative metabolism Fat Browning

MicroRNAs (mirs) Small non-coding RNAs (2-25 nt) Binds to 3 UTR of target mrnas (negative gene regulators) Powerful cellular regulators Known that regulate >1/3 of genomes Aberrant expression in human disease

Elevated mir-34a causes metabolic problems in obesity! Our recent published studies on mir-34a: J. Lee et al., JBC, 21 (SIRT1 in liver) J. Lee and JK Kemper, Review, Aging, 21 T. Fu et al., PNAS, 212 (bkl in liver) SE Choi et al., Aging Cell, 213 (NAMPT in liver) SE Choi and JK Kemper, Review, Mol. and Cells, 213 T. Fu et al., MS in preparation (FGFR1, bkl in adipose) Elevated mir-34a in obesity directly targets βkl in liver

mir-34a may directly target the both members of the FGF21 receptor bkl (a direct mir-34a target) is abundantly expressed in adipose tissue. The 3 UTR of FGFR1 mrna also contains a potential mir-34a site. mir-34a is elevated in adipocyte as well as liver in obese patients. mir-34a 3 UTR of FGFR1 mouse UGUUGGUCGA---UUCUGUGACGGU UCCCCUUGUUGGAC-ACACUGCCU FGF21 FGFR1 βkl Downstream Signaling

Hypothesis Elevated adipocyte mir-34a in obesity contributes to FGF21 resistance by directly targeting the both members of the FGF21 receptor complex, FGFR1 and βkl. If so, downregulation of elevated mir- 34a in obesity should restore FGF21 signaling, which results in beneficial effects on metabolism and energy balance. Obesity FGF21 FGFR1 βkl mir-34a Downstream Signaling ERK/AMPK? Transcription program of fat browning

Inverse correlation between FGFR1/bKL and mir-34a levels in WAT and BAT in obesity ND HFD (16 weeks) Rel mrna level 1.5 ** 1..5. FGFR1 * WAT BAT 1 βkl ** * WAT BAT Rel mirna level 3 2 1 mir-34a * * WAT BAT

mir-34a directly targets the 3 UTR of FGFR1 mrna mir-34a 3 UTR of FGFR1 mouse UGUUGGUCGA---UUCUGUGACGGU UCCCCUUGUUGGAC-ACACUGCCU mir 34a - site Luc FGFR1 3 UTR Overexpression of mir-34a mutated mir-34a site Luc FGFR1 3 UTR Downregulation of mir-34a Rel mirna Level 2 1 plasmids (ng) mir-34a * 5 1 15 2 5 1 5 Rel Luc Activity pcdh-mir-34a 1..5. FGFR1-WT 3 UTR * ** 5 1 15 2 5 1 5 pcdh 1.5 1..5. FGFR1-Mut 3 UTR NS 5 1 15 2 5 1 5 Rel mirna Level 1..5. RNA (nm) mir-34a * ** 5 75 1 5 1 Anti-miR-34a Rel Luc Activity FGFR1-WT 3 UTR 5 4 3 2 1 Anti-Scramble ** 5 75 1 5 1 FGFR1-Mut 3 UTR 2 1 NS 5 75 1 5 1

In vivo role of elevated adipocyte mir-34a in obesity: Downregulation of mir-34a using lenti-anti-mir-34a (L-34ai) injection Inject Lentivirus Sacrifice Days 5d 8d 12d 14d 2d 2 week ND or HFD Start q-rtpcr IB Staining Rel mirna level 5 4 3 2 1 mir-34a * * 2 1 WAT BAT ND (Lenti-E) HFD (Lenti-E) HFD (Lenti-34ai) (g) 6 4 2 ND HFD,L-E HFD,L-34ai Start Body Weight * ** ** ND(L-E) HFD (L-E) HFD(L-34ai) (time) 4wks 2wks 5d 8d 12d 14d 2d Food Intake 3 NS NS NS g / p er d ay 2 1 5d 8d 12d

Downregulation of mir-34a (lenti-anti-mir-34a) reduced adiposity in diet-induced obese mice Types of Adipose Tissue BAT WAT: Visceral Epididymal WAT (ewat): perirenal WAT (prwat) gonadal WAT (gwat) Subcutaneous inguinal WAT (iwat) BAT prwat Adipose Tissue ND(L-E) HFD (L-E) HFD(L-34ai) (g) 1.5 1.5 Adipose Weight * ** ** BAT prwat gwat iwat gwat iwat

Anti-miR-34a in obesity promotes fat browning : UCP1 staining WAT ND(L-E) HFD(L-E) HFD(L-34ai) BAT ND(L-E) HFD(L-E) HFD(L-34ai) UCP1 UCP1 5um 5um 5um 5um 5um 5um UCP1 DAPI UCPI DAPI 5um 5um 5um 5um 5um 5um Adipose Size (um 2 ) 8 *** 6 4 2 ND (Lenti-E) HFD (Lenti-E) HFD (Lenti-34ai) mdna/ndna 1..5. Mitochondrial Number WAT BAT ** 1..5. **

Anti-miR-34a in obesity promotes browning in WAT (beige fat) Visceral ewat prwat He UCPI gwat He UCPI Mitochondrial UCP1 (IHC) ND(L-E) HFD(L-E) HFD(L-34ai) 5um 5um 5um 5um 5um 5um Brown gene expression Rel mrna level Rel mrna level 4 3 2 1 6 4 2 prwat *** * ** Ucp1 Pgc1α Prdm16 ** gwat * Ucp1 Pgc1α Prdm16 Subcutaneous iwat iwat He UCPI 5um 5um 5um Rel mrna level 6 4 2 *** iwat ** * Ucp1 Pgc1α Prdm16

Citrate Synthase (CS) Activity is restored by anti-mir-34a CS catalyzes the first step in the TCA cycle in mitochondria and its activity is a marker for mitochondrial oxidative function. TCA Cycle Citrate Synthase Citrate Synthase Activity (umole/mg/min) gwat * 1 8 6 4 2 C S activity (u m o l/m g/m in ) 8 6 4 2 BAT 1 8 6 4 2 iwat **

Anti-miR-34a in obesity induces true beige fats in all types of WAT: detection of CD137 (Beige fat-specific marker) B UCPI prwat CD137 Merge D UCPI BAT CD137 Merge ND(L-E) ND(L-E) HFD (L-E) HFD (L-34ai) DAPI HFD (L-34ai) HFD (L-E) 5um DAPI 5um

Anti-miR-34a in obesity induces beige fats in all types of WAT: detection of CD137 (Beige fat-specific marker) A UCPI iwat CD137 Merge C UCPI gwat CD137 Merge HFD (L-E) HFD (L-E) ND(L-E) HFD (L-34ai) ND(L-E) DAPI 5um HFD (L-34ai)

Mechanisms? So far, Anti-miR-34a in diet-induced obese mice: reduced adiposity increased mitochondrial number and activity increased UCP1 expression increased browning of WAT (true beige fats) Question: How downregulation of mir-34a in obesity restores FGF21 signaling and promotes fat browning of WAT?

Downregulation of mir-34a restored FGF21 signaling in WAT in vivo 2 week ND or HFD Inject Lentivirus Days 5d 8d 12d 14d 16d Begin FGFR1//βKL Protein ND HFD L-E L-E L-34ai FGF21 - + - + - + βkl FGFR1 1 1.11.63.38.55.53 1.66.16.18.43.62 Kd 15 1 15 1 5 Actin 37 Sacrifice FGF21 (3mins) Collect WAT IB Ac-assay ND HFD L-E L-E L-34ai FGF21 - + - + - + P-Erk T-Erk FGF21 signaling (Erk) 1 4.22 1.4 1.42 2.51 4.9 FGF21 FGFR1 βkl Kd 5 37 5 37 ERK

Downregulation of mir-34a decreased Ac of PGC-1α in WAT in vivo Ac- PGC-1α Ac ND HFD L-E L-E L-34ai FGF21 - + - + - + IP: PGC-1α IB: Acetyl-Lys Kd 1 75 PGC1 Input IB: PGC-1α 1 75 SIRT1 Protein ND HFD L-E L-E L-34ai FGF21 - + - + - + Kd 15 SIRT1 1 1.86.1.12 2.81 2.38 5 Actin 37 mir-34a SIRT1

Both FGF21 signaling and SIRT1 are important for anti-mir-34amediated PGC-1α deac and fat browning in adipocyte sirna Di- 3T3L1 FGF21 βkl sictl siβkl 1.11 IP: βkl IB: βkl B: Tubulin FGFR1 sictl sifgfr1 1.2 IP: FGFR1 IB: FGFR1 B: Tubulin SIRT1 sictl sisirt1 1.27 IB: SIRT1 IB: Tubulin Ac PGC1 A-Sc sictl Anti-miR34a siβkl sifgfr1 FGF21 + + + + + Ac- PGC-1α IP: PGC-1α IB: Acetyl-Lys sisirt1 1 75 1 IB, qpcr Mito#, IHC IB: PGC-1α 75 R el m R N A lev el 2 1 Ucp1 ** *** ** * 2 1 Pgc1α *** *** ** ** * 15 ** ** *** *** ** 1 5 Prdm16 Anti-Sc Anti-SC, FGF21 Anti-34a, FGF21 Anti-34a, FGF21, sifgfr1 Anti-34a, FGF21, siβkl Anti-34a, FGF21, sisirt1

Both FGF21 signaling and SIRT1 are important for anti-mir-34amediated PGC-1α deac and fat browning in adipocyte UCP1 Staining (2x) Anti-Sc Anti-SC,FGF21 Anti-34a,FGF21 3um 3um 3um Anti-34a,siFGFR1,FGF21 Anti-34a,siβKL,FGF21 Anti-34a,siSIRT1,FGF21 3um 3um 3um

Summary FGF21 Obesity FGFR1 βkl Obesity + Anti-miR-34a FGF21 FGFR1 βkl mir-34a ERK/ AMPK? mir-34a ERK/ AMPK? SIRT1 Anti-miR-34a SIRT1 Ac Ac PGC1α PGC1α PGC1α Browning genes (Ucp1, Pgc-1, Prdm16, ----) Directly targets FGFR1 and bkl Impaired FGF21 signaling Directly targets SIRT1 Elevated PGC-1 Ac Dysfunctional Tx activity of PGC-1α Restored FGFR1/βKL/FGF21 signaling Increased SIRT1 Decreased PGC-1 Ac Transcription Program of Beige Fat This study identifies mir-34a a novel target for adipocyte FGF21 signaling, providing exciting potential options for treating obesity-related diseases. Ting Fu et al., MS in preparation

Acknowledgements J. Kim Kemper lab Ting Fu Sunmi Seok, Ph.D. Sung-E Choi, Ph.D. Sam Huang Subodh Kumar, Ph.D. Dong-Hyun Kim, Ph.D. Sangwon Byun, Ph.D. Sanghoon Kwon, Ph.D. Danny Ryerson Erica Yu David Tkac Supported by grants from NIH (DK62777, DK95842), AHA Grant in Aid, and ADA Basic Science Award

Extra energy is stored as FAT (TG) in adipose tissue

Downregulation of mir-34 (Lenti-34ai) reduced TG in both liver and WAT in diet-induced obese mice Oil Red O ND(L-E) HFD (L-E) HFD(L-34ai) 3um 3um 3um 3um 3um 3um

Summary FGF21 Obesity FGFR1 βkl Obesity + Anti-miR-34a FGF21 FGFR1 βkl mir-34a ERK/ AMPK? mir-34a ERK/ AMPK? SIRT1 Anti-miR-34a SIRT1 Ac PGC1α Ac PGC1α PGC1α Browning genes (Ucp1, Pgc-1, Prdm16, ----) Directly targets FGFR1 and bkl Impaired FGF21 signaling Directly targets SIRT1 Elevated PGC-1 Ac Dysfunctional Tx activity of PGC-1α Restored FGFR1/βKL/FGF21 signaling Increased SIRT1 Decreased PGC-1 Ac Transcription Program of Beige Fat Ting Fu et al., MS in preparation

Summary 1. mir-34a directly binds to the 3 UTR of both FGFR1 and bkl mrnas. 2. Elevated adipocyte mir-34a attenuates FGF21 signaling in obesity. 3. Downregulation of mir-34a reduces adiposity in obese mice. 4. Downregulation of mir-34a increases mitochondrial number and function and promotes fat browning (beige fat) in all types of WAT. 5. Mechanisms: Downregulation of mir-34a by lenti-anti-mir-34a Restored FGFR1, bkl, and SIRT1 levels in WAT Restored FGF21 signaling Deacetylation of PGC1-α and increased Tx activity Upregulation of brown genes (UCP1, PGC-1α, PRDM16, ---) Promotes development of Beige Fat in WAT 6. identifies mir-34a a novel target for adipocyte FGF21 signaling, providing exciting potential targets for treating obesity-related diseases. Ting Fu et al., MS in preparation

Downregulation of mir-34 (Lenti-34ai) reduced TG in both liver and WAT in diet-induced obese mice ND(L-E) HFD(L-E) HFD(L-34ai) WAT ORO 1um 1um 1um BAT H&E 1um 1um 1um 1um 1um 1um

Mechanisms? How downregulation of mir-34a in obesity promotes fat browning? Increased FGFR1/βKL levels Restored FGF21 signaling Increased SIRT1 levels Decreased Ac of PGC-1α (a key Tx activator of Mito biogenesis and fat browning) Promotes transcriptional program of fat browning Obesity + Anti-miR-34a FGF21 FGFR1 βkl mir-34a ERK/ AMPK? Anti-miR-34a SIRT1 Ac PGC1α PGC1α Browning genes (Ucp1, Pgc-1, Prdm16, ----)

mir-34a attenuates FGF21 signaling in adipocytes (3T3) R el m ir N A level Overexpression of mir-34a 3 2 1 mir-34a ** Ad- E 34a FGFR1 Ad- E 34a βkl Kd Ad- E 34a 15 1 Kd 15 1 Tubulin Tubulin 5 5 FGF21 signaling FGF21 - + - + P-Erk T-Erk Ad- E 34a Kd 5 37 5 37 1.5 1. NS.5. FGF21 - + - + Ad-E Ad-34a R e l P -E rk /T -E r k P=.13 FGF21 FGFR1 βkl ERK Downregulation of mir-34a Rel mirna level 2 1 mir-34a ** Anti-Sc 34a FGFR1 Anti-Sc anti-mir-34a (nm) 5 15 5 75 1 15 5 75 1 15 IP: FGFR1 IB: FGFR1 Input IB: Tubulin FGF21 signaling Lenti- E 34ai FGF21 - + - + P-Erk T-Erk Kd 5 37 5 37 Rel P-Erk/T-Erk level P=.8 1 5 NS FGF21 - + - + Lenti-E Lenti-34ai

Inverse correlation between FGFR1/bKL and mir-34a levels in obesity ND HFD (16 weeks) Rel mrna level 1.5 ** 1..5. FGFR1 * WAT BAT 1 βkl ** * WAT BAT 3 2 1 WAT FGFR1 βkl Merge BAT FGFR1 βkl Merge HFD ND Rel mirna level mir-34a * * WAT BAT DAPI 5um

However, there are limited amounts of BAT in adult humans, BAT is substantially reduced in obese humans, and more importantly, the physiological significance of BAT in adults has just begun to be appreciated, so it is not clear whether targeting the classical BAT would be a therapeutically effective and ideal approach.

mir-34a hampers Glucose uptake in adipocytes Done by Dr. SungE Choir

ND (Saline) HFD (Oligo) ND Scramble Anti-miR34a H&E 5um 5um 5um Oil Red 5um 5um 5um Glucose (mg/dl) 3 2 1 ND (Saline) HFD (Anti-SC) HFD (Anti-miR-34a) GTT ITT ** ** * ** ** * 15 1 5 15 3 6 12 Time (min) 15 3 6 Time (min)

Increased energy expenditure through uncoupled respiration During mitochondria oxidative phosphorylation pathway, H + leak (which is mediated by UCPs), uncouples the processes of electron transport/proton-gradient generation and ATP synthesis. The energy that is derived from this uncoupling process is released as Heat.

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