The combined partial knockdown of CBS and MPST genes induces inflammation, impairs adipocyte function-related gene expression and disrupts protein persulfidation in human adipocytes

Ci a ion: La o e, J.; A oca, A.; Fe nández-Real, J.M.; Rome o, L.C.; Mo eno-Na a e e, J.M. The Combined Pa ial Knockdown o CBS and MPST Genes Induces In lamma ion, Impai s Adipocy e Func ion-Rela ed Gene Exp ession and Dis up s P o ein Pe sul ida ion in Human Adipocy es. An ioxidan s 2022,11, 1095. h ps://doi.o g/ 10.3390/an iox11061095 Academic Edi o s: Emma Mi idie i and Vincenzo B ancaleone Recei ed: 29 Ma ch 2022 Accep ed: 27 May 2022 Published: 31 May 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). an ioxidan s A icle The Combined Pa ial Knockdown o CBS and MPST Genes Induces In lamma ion, Impai s Adipocy e Func ion-Rela ed Gene Exp ession and Dis up s P o ein Pe sul ida ion in Human Adipocy es Jessica La o e 1,2 , Angeles A oca 3, JoséManuel Fe nández-Real 1,2,4 , Luis C. Rome o 3 and JoséMa ía Mo eno-Na a e e 1,2,* 1 Depa men o Diabe es, Endoc inology and Nu i ion, Ins i u d’In es igacióBiomèdica de Gi ona (IdIBGi), 17190 Sal , Spain; [email p o ec ed]g (J.L.); jm [email p o ec ed] (J.M.F.-R.) 2CIBER Fisiopa ología de la Obesidad y Nu ición (CIBERobn, CB06/03/010), Ins i u o de Salud Ca los III, 28029 Mad id, Spain 3Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones and Uni e sidad de Se illa, 41092 Se ille, Spain; [email p o ec ed] (A.A.); l ome [email p o ec ed] (L.C.R.) 4Depa men o Medicine, Uni e si a de Gi ona, 17003 Gi ona, Spain *Co espondence: jmo [email p o ec ed]g; Tel.: +34-872-987087 (ex . 70) Abs ac : Recen s udies in mice and humans demons a ed he ele ance o H 2 S syn hesising enzymes, such as CTH, CBS, and MPST, in he physiology o adipose issue and he di e en ia ion o p eadipocy e in o adipocy es. He e, ou objec i e was o in es iga e he combined ole o CTH, CBS, and MPST in he p ese a ion o adipocy e p o ein pe sul ida ion and adipogenesis. Combined pa ial CTH,CBS, and MPST gene knockdown was achie ed ea ing ully human adipocy es wi h siRNAs agains hese ansc ip s (siRNA_MIX). Adipocy e p o ein pe sul ida ion was analyzed using label- ee quan i a i e mass spec ome y coupled wi h a dimedone-swi ch me hod o p o ein labeling and pu i ica ion. P o eomic analysis quan i ied 216 p o eins wi h s a is ically di e en le els o pe sul ida ion in KD cells compa ed o con ol adipocy es. In ully di e en ia ed adipocy es, CBS and MPST mRNA and p o ein le els we e abundan , while CTH exp ession was e y low. I is no ewo hy ha siRNA_MIX adminis a ion esul ed in a signi ican dec ease in CBS and MPST exp ession, wi hou impac ing on CTH. The combined pa ial knockdown o he CBS and MPST genes esul ed in educed cellula sul ide le els in pa allel o dec eased exp ession o ele an genes o adipocy e biology, including adipogenesis, mi ochond ial biogenesis, and lipogenesis, bu inc eased p oin lamma o y- and senescence- ela ed genes. I should be no ed ha he combined pa ial knockdown o CBS and MPST genes also led o a signi ican dis up ion in he pe sul ida ion pa e n o he adipocy e p o eins. Al hough among he less pe sul ida ed p o eins, we iden i ied se e al ele an p o eins o adipocy e adipogenesis and unc ion, among he mos pe sul ida ed, key media o s o adipocy e in lamma ion and dys unc ion as well as some p o eins ha migh play a posi i e ole in adipogenesis we e ound. In conclusion, he cu en s udy indica es ha he combined pa ial elimina ion o CBS and MPST (bu no CTH) in adipocy es a ec s he exp ession o genes ela ed o he main enance o adipocy e unc ion and p omo es in lamma ion, possibly by al e ing he pa e n o p o ein pe sul ida ion in hese cells, sugges ing ha hese enzymes we e equi ed o he unc ional main enance o adipocy es. Keywo ds: human adipocy es; adipogenesis; in lamma ion; p o ein pe sul ida ion 1. In oduc ion Func ional adipocy es a e cha ac e ized by an inc eased capaci y o s o e excess uel and p oduce bene icial adipokines, inc eased exp ession o adipogenic genes, bu dec eased exp ession o p oin lamma o y- and cellula senescence- ela ed genes [ 1 – 4 ]. Op imal An ioxidan s 2022,11, 1095. h ps://doi.o g/10.3390/an iox11061095 h ps://www.mdpi.com/jou nal/an ioxidan s An ioxidan s 2022,11, 1095 2 o 14 adipocy e unc ion is equi ed o main ain adipose issue physiology and p e en s obesi y- associa ed me abolic dis u bances [3–6]. Recen s udies in mice and humans demons a ed he ele ance o H 2 S-syn hesising enzymes, such as CTH, CBS, and MPST, o p ese e adipogenesis o adipose issue, heal hy a mass expansion and insulin ac ion [ 7 , 8 ]. These enzymes play a c ucial ole in he di e en ia ion o adipocy es. Speci ically, gene knockdown expe imen s in mouse 3T3-L1 cells and human p eadipocy es demons a ed he ele ance o CTH, CBS and MPST in adipogenesis [7–9]. In e es ingly, inc eased pe sul ida ion in ele an adipogenic p o eins has also been epo ed in human adipocy es [ 8 ]. Since pe sul ida ion o en inc eases he eac i i y o a ge p o eins [ 10 ], inc eased pe sul ida ion in adipogenic enzymes sugges s a possible ole o hese pos - ansla ional modi ica ions in he p omo ion o adipocy e di e en ia ion and in he main enance o adipogenic s a us. Despi e hese in iguing s udies, o he bes o ou knowledge, he combined ole o hese enzymes in he main enance o adipocy e unc ion has no been ye examined. In he p esen s udy, ou objec i e was o in es iga e he combined ole o CTH, CBS, and MPST in he p ese a ion o adipocy e adipogenesis and unc ion and o in es iga e how he combined pa ial knockdown o hese enzymes migh impac he pe sul ida ion o he adipocy e p o eins. 2. Ma e ials and Me hods 2.1. Human P eadipocy e Di e en ia ion and Adipocy e Main enance P ima y human subcu aneous p eadipocy es om a non-diabe ic Caucasian emale wi h BMI < 30 kg/m 2 (Zen-Bio Inc., Resea ch T iangle Pa k, Du ham, NC, USA) we e cul u ed wi h P eadipocy e Medium (PM-1, Zen-Bio Inc.) in a humidi ied incuba o a 37 ◦ C wi h 5% CO 2 . Twen y- ou hou s a e pla ing, cells we e checked o con luence. Once eached, di e en ia ion s a ed wi h comme cially a ailable Di e en ia ion Medium (DM-2, Zen-Bio Inc.) and Main enance Medium (AM-1 Zen-Bio Inc.), ollowing he man- u ac u e ’s ins uc ions. Two weeks a e ini ia ing di e en ia ion, di e en ia ed cells appea ed ounded wi h la ge lipid d ople s appa en in he cy oplasm. The cells we e hen conside ed ma u e adipocy es. Fo wa d siRNA ans ec ions using siRNA_MIX, which included siRNAs agains MPST,CBS, and CTH gene exp ession, we e pe o med a he end o he di e en ia ion p ocess. 2.2. siRNA-Induced Gene Knockdown in Fully Di e en ia ed Adipocy es P ima y human subcu aneous adipocy es we e o wa d ans ec ed wi h siRNAs a he end o di e en ia ion (on day 11). B ie ly, siRNA (Sigma-Ald ich, S . Louis, MO, USA) agains CTH (NM_001190463), CBS (NM_000071), and MPST (NM_001130517) and Lipo- ec amine RNAiMAX (Li e Technologies, Da ms ad , Ge many) we e dilu ed sepa a ely wi h Op i-MEM I Reduced Se um Medium (Li e Technologies, Da ms ad , Ge many) and mixed by pipe ing a e wa ds. The siRNA-RNAiMAX complexes we e le o incuba e o 20 min a oom empe a u e and hen added o he op o he adhe en cells d op-wise. The inal concen a ions o Lipo ec amine RNAiMAX and siRNAs we e 1.6 µ L/cm 2 and 100 nM, espec i ely, in 12-well cell cul u e pla es, and he inal amoun o medium pe well was 1 mL. The ans ec ion condi ions included combined silencing o CTH, CBS and MSPT (100 nM), and a non- a ge ing siRNA (100 nM) o he ehicle. Adipocy es we e ha es ed on day 14 o di e en ia ion, i.e., 72 h a e ans ec ion wi hou changing cell cul u e medium. T ans ec ion e iciency was assessed by eal- ime PCR and Wes e n blo . The MISSION ® siRNAs (Sigma-Ald ich) used we e MISSION ® esiRNA a ge ing human MPST (EHU086511), CBS (EHU099041), and CTH (EHU078251). The MISSION ® siRNA Uni e sal Nega i e Con ol #1 (Sigma-Ald ich, SIC001) was used as a con ol in all expe imen s (siRNA_SCR). An ioxidan s 2022,11, 1095 3 o 14 2.3. Gene Exp ession RNA was ex ac ed om cells using he RNeasy Lipid Tissue Mini Ki (Qiagen, Ge - man own, MD, USA). To al RNA was quan i ied using a spec opho ome e (GeneQuan ; GE Heal h Ca e, Waukesha, WI, USA). RNA was e e se ansc ibed o cDNA using he High-Capaci y cDNA Re e se T ansc ip ion Ki (Li e Technology, Da ms ad , Ge many) acco ding o s anda d p ocedu es. Comme cially a ailable Taq-Man p ime and p obe se s we e used o gene exp ession. Exp ession was assessed by eal- ime PCR using he Ligh Cycle 480 Real-Time PCR Sys em (Roche Diagnos ics, Ba celona, Spain). The comme cially a ailable and p e- alida ed TaqMan ® p ime /p obe se s used we e as ol- lows: Pep idylp olylisome ase A(cyclophilin A)(4333763,PPIA asendogenous con ol),cys a hio- nine γ -lyase (CTH, Hs00542284_m1), cys a hionine β -syn hase (CBS, Hs00163925_m1), me cap- opy u a e sul u ans e ase (MPST, Hs00560401_m1), adiponec in (ADIPOQ, Hs00605917_m1), a yacidbindingp o ein4,adipocy e(FABP4, Hs01086177_m1), pe oxisomep oli e a o -ac i a ed ecep o gamma (PPARG, Hs00234592_m1), pe ilipin 1 (PLIN1, Hs00160173_m1), PPARG coac i a- o 1alpha(PPARGC1A,Hs00173304_m1), a yacidsyn hase(FASN,Hs00188012_m1),ace yl-CoA ca boxylase alpha (ACACA, Hs00167385_m1), solu e ca ie amily 2 membe 4 (SLC2A4 o GLUT4, Hs00168966_m1), in e leukin 6 (in e e on, be a 2) (IL6, Hs00985639_m1), umo nec osis ac o (TNF, Hs00174128_m1), C-X-C mo i chemokine ligand 8 (CXCL8 o IL8, Hs00174103_m1), C–C mo i chemokine ligand 2 (CCL2 o MCP1, Hs00234140_m1), and umo p o ein p53 (TP53, Hs01034249_m1). 2.4. Wes e n Blo The p o eins we e ex ac ed om cells using adioimmunop ecipi a ion assay (RIPA) bu e (0.1% SDS, 0.5% sodium deoxy-chola e, 1% Nonide P-40, 150 mmol/L NaCl, and 50 mmol/L T is-HCl, pH 8.0) supplemen ed wi h a comme cially a ailable cock ail o p o ease inhibi o s (Millipo e, Sigma, Mad id, Spain). Cellula deb is and lipids we e elimina ed by cen i uga ion o he solubilized samples a 12,000 × g o 10 min a 4 ◦ C, e- co e ing he soluble ac ion. The p o ein concen a ion was de e mined using he RC/DC P o ein Assay (Bio-Rad Labo a o ies, He cules, CA, USA). P o ein ex ac s we e p ocessed in SDS-PAGE and ans e ed o ni ocellulose memb anes by con en ional p ocedu es. A e blocking wi h 5% BSA in TBS-Tween, he memb anes we e incuba ed wi h p ima y an ibodies, ollowed by incuba ion wi h ho se adish pe oxidase conjuga ed goa an i- abbi an ibody (Cell Signaling, 7074). P o ein signal was de ec ed by chemiluminescence Chemi- Doc ™ Gel Imaging Sys em (Bio-Rad, Cali o nia, USA). Ac in was used as a housekeeping gene-coded p o ein. All p ima y an ibodies we e used a 1:1000 dilu ions and we e he ol- lowing: CBS (sc-133154, San a C uz Bio echnology, San a C uz, CA, USA), CTH (sc-365382, San a C uz Bio echnology), MPST (sc-376168, San a C uz Bio echnology), FAS (C20G5, Cell Signaling), and β-ac in (sc-47778, San a C uz Bio echnology). 2.5. Quan i ica ion o Hyd ogen Sul ide in Biological Ma ices by LC-MS/MS Hyd ogen sul ide was quan i ied ollowing a p e iously desc ibed me hod [ 11 ]. A 50 µ L sample (cell lysa e) was spiked wi h 70 µ L o MBB solu ion (20 mM). The mix u e was o exed, incuba ed o 60 min a oom empe a u e, and he de i a iza ion eac ion s opped by adding 10 µ L 20% o mic acid. The mix u e was subjec ed o cen i uga ion a 12,000 × pm o 10 min. The de i a i e sul ide dibimane was analyzed by LC-MS/MS using he ExionLC ™ UPLC sys em (Sciex, F amingham, MA, USA) wi h a e e sed-phase column (100 mm × 4.6 mm × 100 Å pa icle, Kine ex XB-C18, Phenomenex, To ance, CA, USA). The mobile phases we e 0.1% o mic acid in H 2 O (Bu e A, HPLC/LCMS g ade) and 0.1% o mic acid in ace oni ile (Bu e B, HPLC/LC-MS g ade). A 5 µ L sample was loaded pe injec ion, and he g adien , applied a a low a e o 600 µ L min −1 , was as ollows: 1.7 min 80% A, om 1.7 o 3 min linea g adien om 80% A o 20% A, 1 min isoc a ic 20% A, 1 min linea g adien om 20% A o 80% A, and hold 3 min a 80% A o e-equilib a e he column. Mass spec a we e acqui ed using a QTRAP 6500 + iple quad upole (Sciex, F amingham, MA, USA) equipped wi h an elec osp ay ioniza ion sou ce ope a ing in An ioxidan s 2022,11, 1095 4 o 14 nega i e ioniza ion mode using an ion sp ay ol age o − 4500 V. The o he ESI pa ame e s we e as ollows: cu ain gas, 35 psi; collision gas, medium; empe a u e, 500 ◦ C; nebulize gas (GS1), 60 psi; and hea e gas (GS2), 60 psi. Da a we e acqui ed wi h Analys ® 1.7 so wa e in he mul iple eac ion moni o ing (MRM) mode wi h a de ec ion window o 60 s . The measu ed ioniza ion adduc s [M-H] − selec ed o iden i ica ion and quan i ica ion we e 412.9 m/z (Q1) and 190.9 m/z (Q3) and op imized declus e ing po en ial ( DP −45 V ) and collision ene gy (CE − 24 V). Da a we e p ocessed wi h Sciex OS ® so wa e (Sciex, Redwood Ci y, CA, USA) o peak in eg a ion and quan i ica ion. 2.6. P o ein Pe sul ida ion A o al o 500 µ g o p o eins om adipocy e lysa es in 50 mM T is-HCl, pH 8, supple- men ed wi h 1% p o ease inhibi o (cOmple e ™ , SigmaAld ich) and 2% SDS was incuba ed wi h 5 mM 4-chlo o-7-ni obenzo u azan (Cl-NBF) a 37 ◦ C o 30 min, p o ec ed om ligh . A me hanol/chlo o o m p ecipi a ion was pe o med o elimina e excess Cl-NBF, and p o ein pelle s ob ained we e washed wi h cold me hanol, d ied, and dissol ed in 50 mM T is-HCl, pH 8, wi h 2% SDS, as p e iously desc ibed [ 12 , 13 ]. The p o eins we e hen incuba ed wi h 100 µ M DCP-Bio1 a 37 ◦ C o 1.5 h. A e wa ds, p o eins we e p ecip- i a ed wi h me hanol/chlo o o m and dissol ed in 50 mM T is-HCl, pH 8, supplemen ed wi h 0.1% SDS. P o eins we e incuba ed wi h Se a-Mag ™ Magne ic S ep a idin (Cy i a, Mad id, Spain) a 4 ◦ C o e nigh wi h agi a ion and hen, beads we e washed wi h 8 col- umn olumes o 50 mM T is-HCl, pH 8, wi h 0.001% Tween, 2 column olumes o 50 mM T is-HCl, pH 8, and 1 column olume o pu e HPLC-quali y wa e . A e washing, beads we e incuba ed 5 h wi h agi a ion wi h 2.25 M ammonium hyd oxide a RT. The sample was hen neu alized wi h o mic acid and he p o ein concen a ion was de e mined. A o al o 50 µ g o p o eins we e ypsinized (GOLD, P omega) ollowing he il e -aided sample p epa a ion (FASP) me hod [14]. T ypsin was added o a ypsin:p o ein a io o 1:20, and he mix u e was incuba ed o e nigh a 37 ◦ C, d ied ou in a RVC2 25 speed ac concen a o (Ch is ), and esuspended in 0.1% FA. Pep ides we e desal ed and esuspended in 0.1% FA using C18 s age ips (Millipo e). The samples we e analyzed in a imsTOF P o wi h PASEF (B uke Dal onics, Bille ica, MA, USA) coupled online o an E osep ONE liquid ch oma og aph (E osep). Hence, 200 ng we e di ec ly loaded on o he E osep ONE and esol ed using he 30 samples-pe -day p o ocol. The p ocessed da a we e analyzed wi h he MQ (Max Quan ) sea ch engine and he label- ee quan i ica ion was pe o med using PEAKS S udio (BSI, Mississauga, Canada) [ 15 ]. P o ein iden i ica ion and quan i ica ion we e ca ied ou using PEAKS X so wa e (Bioin o ma ics solu ions). The sea ches we e ca - ied ou agains a da abase consis ing o Homo sapiens (Unip o /Swissp o ), wi h p ecu so and agmen ole ances o 20 ppm and 0.05 Da. Only p o eins iden i ied wi h a leas wo pep ides a FDR < 1% we e conside ed o u he analysis. P o ein abundances in e ed om PEAKS we e loaded on o Pe seus compu a ional pla o m [ 16 ], log2 ans o med, and impu a ed. A - es was used o add ess signi ican di e ences in p o ein abundances wi hin each sample g oup unde analysis. The mass spec ome y p o eomics da a ha e been deposi ed in he P o eomeXchange Conso ium [ 17 ] ia he PRIDE pa ne eposi o y wi h he da ase iden i ie PXD032370 and 10.6019/PXD032370. Func ional p o ein associa ion ne wo ks we e explo ed using he STRING da abase (STRING: unc ional p o ein associa ion ne wo ks (s ing-db.o g) access da e 26 Feb ua y 2022). 2.7. S a is ical Analysis S a is ical analyses we e pe o med using SPSS 12.0 so wa e. The unpai ed - es and nonpa ame ic es (Mann–Whi ney es ) we e used o analyze in i o expe imen al da a. The le els o s a is ical signi icance we e se a p< 0.05. An ioxidan s 2022,11, 1095 5 o 14 3. Resul s and Discussion 3.1. The Rele ance o CTH, CBS and MPST on Human Adipocy es In ully di e en ia ed adipocy es, CBS and MPST mRNA and p o ein le els we e abundan , whe eas CTH exp ession was e y low (Figu e 1A,B). I should be no ed ha siRNA_MIX adminis a ion esul ed in a signi ican dec ease in CBS and MPST exp es- sion, wi hou impac ing on CTH (Figu e 1A,B). Possibly, he e y low ansc ip ion and u no e o CTH make i esis an o he e ec o siRNA. The combined pa ial knock- down o CBS and MPST genes led o a signi ican educ ion in cellula sul ide le els (Figu e 1C), indica ing ha H 2 S biosyn hesis was a enua ed in hese cells. In e es ingly, he combined pa ial CBS and MPST gene knockdown also esul ed in dec eased exp es- sion o ele an genes o adipocy e biology, including adipogenesis (ADIPOQ, FABP4, PLIN1, SLC2A4, PPARG), mi ochond ial biogenesis (PPARGC1A), and lipogenesis (ACACA and FASN mRNA, and FAS p o ein) (Figu e 1D–L), bu inc eased p oin lamma o y- and senescence- ela ed genes (TP53) (Figu e 1M–Q). Impo an ly, hese da a indica ed ha MPST and CBS we e no only equi ed du ing adipocy e di e en ia ion o mouse 3T3- L1 cells [ 9 ] o human p eadipocy es [ 8 ], bu hese p o eins we e also necessa y o he unc ional main enance o adipocy es. In con as o ha obse ed du ing adipocy e di e - en ia ion o in mouse 3T3-L1 adipocy es [ 7 , 18 , 19 ], in ully di e en ia ed human adipocy es, he exp ession o CTH was much lowe han CBS and MPST. An ioxidan s 2022, 11, x FOR PEER REVIEW 5 o 15 2.7. S a is ical Analysis S a is ical analyses we e pe o med using SPSS 12.0 so wa e. The unpai ed - es and nonpa ame ic es (Mann–Whi ney es ) we e used o analyze in i o expe imen al da a. The le els o s a is ical signi icance we e se a p < 0.05. 3. Resul s and Discussion 3.1. The Rele ance o CTH, CBS and MPST on Human Adipocy es In ully di e en ia ed adipocy es, CBS and MPST mRNA and p o ein le els we e abundan , whe eas CTH exp ession was e y low (Figu e 1A,B). I should be no ed ha siRNA_MIX adminis a ion esul ed in a signi ican dec ease in CBS and MPST exp es- sion, wi hou impac ing on CTH (Figu e 1A,B). Possibly, he e y low ansc ip ion and u no e o CTH make i esis an o he e ec o siRNA. The combined pa ial knock- down o CBS and MPST genes led o a signi ican educ ion in cellula sul ide le els (Fig- u e 1C), indica ing ha H2S biosyn hesis was a enua ed in hese cells. In e es ingly, he combined pa ial CBS and MPST gene knockdown also esul ed in dec eased exp ession o ele an genes o adipocy e biology, including adipogenesis (ADIPOQ, FABP4, PLIN1, SLC2A4, PPARG), mi ochond ial biogenesis (PPARGC1A), and lipogenesis (ACACA and FASN mRNA, and FAS p o ein) (Figu e 1D–L), bu inc eased p oin lamma o y- and se- nescence- ela ed genes (TP53) (Figu e 1M–Q). Impo an ly, hese da a indica ed ha MPST and CBS we e no only equi ed du ing adipocy e di e en ia ion o mouse 3T3-L1 cells [9] o human p eadipocy es [8], bu hese p o eins we e also necessa y o he unc- ional main enance o adipocy es. In con as o ha obse ed du ing adipocy e di e en- ia ion o in mouse 3T3-L1 adipocy es [7,18,19], in ully di e en ia ed human adipocy es, he exp ession o CTH was much lowe han CBS and MPST. No isual di e ences in lipid d ople s we e obse ed be ween con ol and combined pa ial knockdown cells (da a no shown). Conside ing ha adipocy e dedi e en ia ion becomes mo phologically e iden a leas one week a e he s a o he p ocess [20], longe gene knockdown expe imen s should be equi ed o app ecia e signi ican changes in in acellula lipid accumula ion. Figu e 1. The impac o he combined pa ial knockdown o CBS and MPST gene on ele an genes o adipocy e biology. The e ec o siRNA_MIX adminis a ion on CBS,CTH and MPST mRNA ( A ) and p o ein ( B ) le els, on in acellula sul ide le els ( C ), and on exp ession o adipogenic (ADIPOQ, FABP4, PLIN1, SLC2A4, PPARG, PPARGC1A)-, lipogenic (ACACA, FASN)-, p oin lamma o y (CCL2, IL6, IL8, TNF)- and cellula senescence (TP53)- ela ed genes and on FAS p o ein le els ( D – Q ). *p< 0.05 , ** p< 0.01 and *** p< 0.001 compa ed o siRNA_SCR. No isual di e ences in lipid d ople s we e obse ed be ween con ol and combined pa ial knockdown cells (da a no shown). Conside ing ha adipocy e dedi e en ia ion becomes mo phologically e iden a leas one week a e he s a o he p ocess [ 20 ], longe gene knockdown expe imen s should be equi ed o app ecia e signi ican changes in in acellula lipid accumula ion. An ioxidan s 2022,11, 1095 6 o 14 3.2. Impac o he Combined Pa ial Knockdown o MPST and CBS Genes on Adipocy e P o ein Pe sul ida ion The possible link be ween CTH, CBS, and MPST ac i i ies and p o ein pe sul ida ion du ing human adipocy e di e en ia ion [ 8 ] sugges s ha hese enzymes migh modula e pe sul ida ion in se e al adipogenic p o eins, a ec ing he physiology o adipocy es. To es his hypo hesis, we examined whe he he educ ion o adipogenic ma ke s obse ed in human adipocy es wi h he combined pa ial knockdown o MPST and CBS genes was linked o al e ed pe sul ida ion in key adipogenic p o eins. Fo his pu pose, we used a mass spec ome y label- ee quan i a i e p o eomic app oach combined wi h he dimedone-swi ch me hod o measu e he p o ein p o ile o pe sul ida ion. P o eins om h ee biological eplica es om ully di e en ia ed human adipocy es, con ol, and he combined pa ial MPST and CBS gene knockdown we e isola ed and subjec ed o he dimedone-swi ch p ocedu e o isola ion o he pe sul ida ed p o eins. The p o eomic analysis quan i ied 1834 p o eins wi h a FDR h eshold o 1% (Supplemen a y Da ase S1) o which 216 showed s a is ically di e en le els o pe sul ida ion in KD cells compa ed o con ol adipocy es (S uden - es , p< 0.05) (Supplemen a y Da ase S2). F om hese p o eins, 136 p o eins we e less pe sul ida ed in KD cells, and 80 p o eins we e mo e pe sul ida ed. When unc ional p o ein associa ion ne wo ks we e explo ed using he STRING da abase, we ound ha hose di e en ially less pe sul ida ed p o eins in human adipocy es wi h he combined pa ial knockdown o MPST and CBS genes we e associa ed wi h ele- an me abolic p ocesses o adipocy es. These p ocesses included he biosyn he ic p ocess o ace yl-CoA and acyl-CoA, ica boxylic acid cycle, he me abolic p ocess o py u a e and glucose, ex acellula ma ix o ganiza ion, he egula ion o he mRNA s abili y, me abolic p ocess o sul u compound, he oxida ion- educ ion p ocess, and he egula ion o ca abolic p ocess (FDR < 0.02, Figu e 2A). O he wise, di e en ially inc eased pe sul ida ed p o eins in human adipocy es wi h he combined pa ial knockdown o MPST and CBS genes we e mainly associa ed wi h p ocesses ela ed o immune esponse- and cy okine-media ed signaling pa hways (FDR < 0.005, Figu e 2B). An ioxidan s 2022, 11, x FOR PEER REVIEW 7 o 15 Figu e 2. P o eomic pe sul ida ion ne wo ks analysis. Analysis o unc ional p o ein associa ion ne - wo ks using STRING da abase o di e en ially dec eased (A) and inc eased (B) pe sul ida ed p o- eins in siRNA_MIX- compa ed o siRNA_SCR- ea ed cells. FDR, alse disco e y a e adjus ed p- alue. Speci ically, among hose p o eins ound less pe sul ida ed, se e al ele an p o eins o he main enance o adipocy e adipogenesis and unc ion we e iden i ied, including RAB4A, IDH2, ITGA5, NPC1, ACO1, LRP1, AHNAK, ARPC3, ADH1B, ALDH1A3, LGALS1, PHB, COL6A3, PLIN3, FABP4, THBS1, ANXA2, PLIN4, ACSL1, ANXA1, ELAVL1, ALDH1A1, and HNRNPA1 (Figu e 3A, all wi h p < 0.05). P e ious s udies sup- po ed he ele ance o hese p o eins in adipocy e biology. RAB4A is a small GTPase ha pa icipa es in adipocy e GLUT4 a icking, exe ing an impo an ole in glucose up ake [21,22]. IDH2 a enua es adipocy e in lamma ion h ough he induc ion o α-ke oglu a a e p oduc ion [23] and is equi ed o lipogenesis in hese cells [24]. ITGA5 p omo es ib osis o adipose issue [25] and inhibi s adipocy e di e en ia ion in human adipose issue-de- i ed s em cells [26]. Dec eased exp ession o he NPC1 gene o he pa ial dele ion o his gene p omo ed weigh gain and a mass expansion [27–29], sugges ing a ele an ole o NPC1 in he p e en ion o obesi y and adipocy e hype ophy, possibly inc easing lipol- ysis and be a oxida ion and dec easing lipogenesis and iglyce ide accumula ion in adi- pocy es [30]. NPC1 is inc eased in adipocy es[31], bu i was no associa ed wi h adipo- genesis [32]. ACO1 is impo an o main ain adipogenesis in adipocy es, possibly modu- la ing cy osolic NADPH le els and in acellula i on homeos asis, bo h p ocesses equi ed o adipocy e biology [33]. LRP1 is equi ed o adipocy e di e en ia ion and o main ain adipocy e adipogenesis [34], possibly in pa due o i s in e ac ion wi h ShcA [35], o ac - ing as APOA4 ecep o , and a o ing APOA4-induced glucose up ake [36]. Se e al s ud- ies ha e demons a ed ha AHNAK is equi ed o adipocy e di e en ia ion [37–39]. Mechanis ically, a ecen s udy demons a ed ha AHNAK gene knockdown dec eased adipogenesis h ough he supp ession o Bmp 1α exp ession and dec eased BMP4/ Bmp 1α signaling [39]. ARPC3 is ano he p o ein equi ed o adipocy e di e en ia ion, exe ing a ele an ole in he la e s age o he p ocess, pa icipa ing in GLUT4 exocy osis and insulin signal ansduc ion, and sugges ing a ele an ole in main aining insulin ac- ion in adipocy es [40]. ADH1B modula es in acellula lipid accumula ion, wi hou im- pac ing adipogenesis [41], and p ese ed insulin-s imula ed glucose up ake in adipocy es Figu e 2. P o eomic pe sul ida ion ne wo ks analysis. Analysis o unc ional p o ein associa ion ne - wo ks using STRING da abase o di e en ially dec eased ( A ) and inc eased ( B ) pe sul ida ed p o eins in siRNA_MIX- compa ed o siRNA_SCR- ea ed cells. FDR, alse disco e y a e adjus ed p- alue. An ioxidan s 2022,11, 1095 7 o 14 Speci ically, among hose p o eins ound less pe sul ida ed, se e al ele an p o- eins o he main enance o adipocy e adipogenesis and unc ion we e iden i ied, includ- ing RAB4A, IDH2, ITGA5, NPC1, ACO1, LRP1, AHNAK, ARPC3, ADH1B, ALDH1A3, LGALS1, PHB, COL6A3, PLIN3, FABP4, THBS1, ANXA2, PLIN4, ACSL1, ANXA1, ELAVL1, ALDH1A1, and HNRNPA1 (Figu e 3A, all wi h p< 0.05). P e ious s udies suppo ed he ele ance o hese p o eins in adipocy e biology. RAB4A is a small GTPase ha pa ici- pa es in adipocy e GLUT4 a icking, exe ing an impo an ole in glucose up ake [ 21 , 22 ]. IDH2 a enua es adipocy e in lamma ion h ough he induc ion o α -ke oglu a a e p o- duc ion [ 23 ] and is equi ed o lipogenesis in hese cells [ 24 ]. ITGA5 p omo es ib osis o adipose issue [ 25 ] and inhibi s adipocy e di e en ia ion in human adipose issue-de i ed s em cells [ 26 ]. Dec eased exp ession o he NPC1 gene o he pa ial dele ion o his gene p omo ed weigh gain and a mass expansion [ 27 – 29 ], sugges ing a ele an ole o NPC1 in he p e en ion o obesi y and adipocy e hype ophy, possibly inc easing lipolysis and be a oxida ion and dec easing lipogenesis and iglyce ide accumula ion in adipocy es [ 30 ]. NPC1 is inc eased in adipocy es [ 31 ], bu i was no associa ed wi h adipogenesis [ 32 ]. ACO1 is impo an o main ain adipogenesis in adipocy es, possibly modula ing cy osolic NADPH le els and in acellula i on homeos asis, bo h p ocesses equi ed o adipocy e biology [ 33 ]. LRP1 is equi ed o adipocy e di e en ia ion and o main ain adipocy e adipogenesis [ 34 ], possibly in pa due o i s in e ac ion wi h ShcA [ 35 ], o ac ing as APOA4 ecep o , and a o ing APOA4-induced glucose up ake [ 36 ]. Se e al s udies ha e demons a ed ha AHNAK is equi ed o adipocy e di e en ia ion [ 37 – 39 ]. Mechanis ically, a ecen s udy demons a ed ha AHNAK gene knockdown dec eased adi- pogenesis h ough he supp ession o Bmp 1αexp ession and dec eased BMP4/ Bmp 1α signaling [ 39 ]. ARPC3 is ano he p o ein equi ed o adipocy e di e en ia ion, exe ing a ele an ole in he la e s age o he p ocess, pa icipa ing in GLUT4 exocy osis and insulin signal ansduc ion, and sugges ing a ele an ole in main aining insulin ac ion in adipocy es [ 40 ]. ADH1B modula es in acellula lipid accumula ion, wi hou impac ing adipogenesis [ 41 ], and p ese ed insulin-s imula ed glucose up ake in adipocy es [ 42 ]. Inhibi ion o ALDH1A1 educed isce al a [ 43 ], nega i ely impac ing adipogenesis in isce al adipose issue [ 44 ]. LGALS1 (galec in 1) is an adipocy e sec e ed ac o [ 45 ] ha enhances he ansc ip ional ac i i y o PPARG, inc easing adipogenesis and accumula ion o lipids in adipocy es [ 46 ]. Gain–loss in i o s udies demons a ed ha his p o ein is c ucial o adipose issue expansion and adiposi y [ 46 ]. LGALS1 exp ession in subcu a- neous adipose issue has been iden i ied as a ele an ac o o con e he isk o weigh egain [ 47 ]. Pha macological inhibi ion o LGALS1 displayed impo an an i-obesogenic and an i-adipogenic e ec s [ 48 , 49 ], s eng hening he ole o his p o ein in he de elopmen o a mass and obesi y. O he wise, ci cula ing galec in-1 le els we e nega i ely associa ed wi h ype 2 diabe es [ 50 , 51 ], indica ing ha i migh be associa ed wi h heal hy adiposi y. PHB is induced du ing adipocy e di e en ia ion, in esponse o insulin and PPAR γ agonis , and i s o e exp ession in 3T3-L1 ib oblas s was su icien o induce adipogenesis [ 52 ], bu i s gene knockdown educes he exp ession o adipogenic genes and lipid accumula ion and impai s mi ochond ial dys unc ion [ 53 , 54 ]. In adipocy es, p ohibi in is equi ed o a y acid up ake [ 55 ]. COL6A3 gene knockdown inc eased he iglyce ide con en , lipolysis, insulin-induced Ak phospho yla ion, and he exp ession o adipogenic genes (pe oxisome p oli e a o -ac i a ed ecep o - γ , glucose anspo e , adiponec in, and a y acid binding p o ein), indica ing imp o ed adipocy e unc ion and insulin sensi i i y [ 56 ]. In addi ion, COL6A3 knockdown also dec eased basal adipocy e MCP1 mRNA exp ession, educed sec e ed p o ein le els, and a enua ed TNF α - and LPS-induced MCP1 gene exp ession [ 57 ]. In con as , inc eased COL6A3 mRNA in adipocy es is associa ed wi h obesi y, insulin esis ance, and in lamma ion o adipose issue [ 58 , 59 ], suppo ing he nega i e impac o his p o ein on adipocy es and adipose issue. Annexin A1 (AnxA1) is an endogenous glucoco icoid egula ed p o ein ha modula es sys emic an i-in lamma o y p ocesses. AnxA1 gene exp ession and p o ein we e signi ican ly up- egula ed du ing adipogenesis in human SGBS p eadipocy es [ 60 ]. Epididymal a mass was educed by ANXA1 gene An ioxidan s 2022,11, 1095 8 o 14 dele ion, bu adipocy e size was unchanged, sugges ing ha ANXA1 is equi ed o main- aining adipocy e cell numbe [ 61 ]. Fu he mo e, he p og essi e accumula ion o Annexin A2 (AnxA2) in he myo ibe ma ix causes muscle- esiden ib o/adipogenic p ecu so s (FAP) di e en ia ion in o adipocy es, and deple ion o AnxA2 p e en s FAP adipogenesis and muscle loss [ 62 ]. ACSL1 is equi ed o be a oxida ion in adipocy es, and i is associ- a ed o adipose insulin sensi i i y, adipogenesis and adipocy e a y acid up ake [ 63 – 70 ]. ELAVL1 (also named HuR) is an impo an ep esso o adipogenesis. Knockdown and o e exp ession o HuR in p ima y adipocy e cul u e enhances and inhibi s adipogenesis, espec i ely [ 71 ]. HuR also exe s an impo an ole in he p e en ion o adipocy e hype - ophy and in lamma ion o adipose issue [ 72 ]. Howe e , p e ious s udies in 3T3-L1 cells indica e ha HuR is equi ed in ea ly e en s o adipogenesis, allowing C/EBP β mRNA ansloca ion in o he cy osol, and i s p ope ansla ion, a p ocess equi ed o ac i a e CEBP α and PPAR γ in mi o ic clonal expansion [ 73 , 74 ]. Impo an ly, he educ ion in HuR pe sul ida ion, esul ed in he s abiliza ion o HuR- a ge mRNAs, and inc eased i s ans- la ion and exp ession [ 75 ], sugges ing ha he dec ease in HuR pe sul ida ion imp o ed HuR dime iza ion and i s ac i i y, and consequen ly, i has nega i e e ec s on adipocy e adipogenesis. In e es ingly, pe ilipin pe sul ida ion inhibi s lipid mobiliza ion and lipolysis in adipocy es, and con ibu es o adequa e main enance o lipid s o age [ 76 ]. HNRNPA1 is a known egula o o INSR exon 11 splicing, which could ha e an impac on adipose insulin ac ion [77]. An ioxidan s 2022, 11, x FOR PEER REVIEW 9 o 15 Figu e 3. Ra e o pe sul ida ion o ele an p o eins in ol ed in adipocy e unc ion and he main e- nance o adipogenesis. Le el o p o ein pe sul ida ion in siRNA_MIX samples (black boxes, in pe - cen age) compa e o he le el in con ol samples (g ey boxes). (A) ACO1, Cy oplasmic aconi a e hyd a ase; ACSL1, Long-chain- a y-acid—CoA ligase 1; ADH1B, All- ans- e inol dehyd ogenase [NAD(+)] ADH1B; AHNAK, Neu oblas di e en ia ion-associa ed p o ein AHNAK; ALDH1A1, Re inal dehyd ogenase 1; ALDH1A3, Aldehyde dehyd ogenase amily 1 membe A3; ANXA1, An- nexin A1; ANXA2, Annexin A2; ARPC3, Ac in- ela ed p o ein 2/3 complex subuni 3; COL6A3, Col- lagen alpha-3(VI) chain; ELAVL1, ELAV-like p o ein 1; FABP4, Fa y acid-binding p o ein adipo- cy e; HNRNPA1, He e ogeneous nuclea ibonucleop o ein A1; IDH2, Isoci a e dehyd ogenase [NADP] mi ochond ial; ITGA5, In eg in alpha-5; LGALS1, Galec in-1; LRP1, Low-densi y lipop o- ein ecep o - ela ed p o ein 1; NPC1, NPC in acellula choles e ol anspo e 1; PHB, P ohibi in; PLIN3, Pe ilipin-3; PLIN4, Pe ilipin-4;RAB4A, Ras- ela ed p o ein Rab-4A; THBS1, Th ombospon- din-1. (B) EXOC7, Exocys complex componen 7; IFI16, Gamma-in e e on-inducible p o ein 16; MAP4K4, Mi ogen-ac i a ed p o ein kinase kinase kinase kinase 4; PREB, P olac in egula o y ele- men -binding p o ein; RELA, T ansc ip ion ac o p65; SLC39A14, Me al ca ion sympo e ZIP14; STAT1, Signal ansduce and ac i a o o ansc ip ion 1-alpha/be a; STAT3, Signal ansduce and ac i a o o ansc ip ion 3. S uden - es signi icance: * p < 0.05; ** p < 0.01. Among he mos pe sul ida ed p o eins, some key media o s o adipocy e in lamma- ion and dys unc ion (IFI16, STAT3, RELA, STAT1, MAP4K4, PREB) we e ound, bu also p o eins ha migh ha e a posi i e ole in adipogenesis (EXOC7, SLC39A14) (Figu e 3B, all wi h p < 0.05). In mice and humans, inc eased IFI16 le els a e associa ed wi h la ge adipocy es, enhanced in lamma o y s a e, and impai ed insulin-s imula ed glucose up- ake in whi e adipose issue [78]. STAT3 is a ele an ansc ip ion ac o o p oin lamma- o y genes in adipocy es ha is associa ed wi h adipocy e in lamma ion [79,80]. RELA is a ele an ansc ip ion ac o associa ed wi h adipocy e in lamma ion [81] ha inhibi s adipogenesis and accumula ion o lipids in adipocy es [82]. The ac i a ion o STAT1 an- sc ip ion ac o inhibi s adipogenesis and p omo es adipocy e and adipose issue in lam- ma ion [83–88]. Isola ed ma u e adipocy es om obese indi iduals had inc eased exp es- sion o mi ogen-ac i a ed p o ein 4 kinase 4 (MAP4K4), which is known o inhibi PPARγ ansc ip ional ac i i y, adipogenesis, and insulin-s imula ed glucose anspo [89,90]. In Figu e 3. Ra e o pe sul ida ion o ele an p o eins in ol ed in adipocy e unc ion and he main- enance o adipogenesis. Le el o p o ein pe sul ida ion in siRNA_MIX samples (black boxes, in pe cen age) compa e o he le el in con ol samples (g ey boxes). ( A ) ACO1, Cy oplasmic aconi a e hyd a ase; ACSL1, Long-chain- a y-acid—CoA ligase 1; ADH1B, All- ans- e inol dehyd ogenase [NAD(+)] ADH1B; AHNAK, Neu oblas di e en ia ion-associa ed p o ein AHNAK; ALDH1A1, Re i- nal dehyd ogenase 1; ALDH1A3, Aldehyde dehyd ogenase amily 1 membe A3; ANXA1, Annexin A1; ANXA2, Annexin A2; ARPC3, Ac in- ela ed p o ein 2/3 complex subuni 3; COL6A3, Colla- gen alpha-3(VI) chain; ELAVL1, ELAV-like p o ein 1; FABP4, Fa y acid-binding p o ein adipocy e; HNRNPA1, He e ogeneous nuclea ibonucleop o ein A1; IDH2, Isoci a e dehyd ogenase [NADP] mi ochond ial; ITGA5, In eg in alpha-5; LGALS1, Galec in-1; LRP1, Low-densi y lipop o ein ecep o - ela ed p o ein 1; NPC1, NPC in acellula choles e ol anspo e 1; PHB, P ohibi in; PLIN3, Pe ilipin- 3; PLIN4, Pe ilipin-4;RAB4A, Ras- ela ed p o ein Rab-4A; THBS1, Th ombospondin-1. ( B ) EXOC7, Exocys complex componen 7; IFI16, Gamma-in e e on-inducible p o ein 16; MAP4K4, Mi ogen- ac i a ed p o ein kinase kinase kinase kinase 4; PREB, P olac in egula o y elemen -binding p o ein; RELA, T ansc ip ion ac o p65; SLC39A14, Me al ca ion sympo e ZIP14; STAT1, Signal ansduce and ac i a o o ansc ip ion 1-alpha/be a; STAT3, Signal ansduce and ac i a o o ansc ip ion 3. S uden - es signi icance: * p< 0.05; ** p< 0.01. An ioxidan s 2022,11, 1095 9 o 14 Among he mos pe sul ida ed p o eins, some key media o s o adipocy e in lamma- ion and dys unc ion (IFI16, STAT3, RELA, STAT1, MAP4K4, PREB) we e ound, bu also p o eins ha migh ha e a posi i e ole in adipogenesis (EXOC7, SLC39A14) ( Figu e 3B, all wi h p< 0.05). In mice and humans, inc eased IFI16 le els a e associa ed wi h la ge adipocy es, enhanced in lamma o y s a e, and impai ed insulin-s imula ed glucose up ake in whi e adipose issue [ 78 ]. STAT3 is a ele an ansc ip ion ac o o p oin lamma o y genes in adipocy es ha is associa ed wi h adipocy e in lamma ion [ 79 , 80 ]. RELA is a ele an ansc ip ion ac o associa ed wi h adipocy e in lamma ion [ 81 ] ha inhibi s adipogenesis and accumula ion o lipids in adipocy es [ 82 ]. The ac i a ion o STAT1 an- sc ip ion ac o inhibi s adipogenesis and p omo es adipocy e and adipose issue in lamma- ion [ 83 – 88 ]. Isola ed ma u e adipocy es om obese indi iduals had inc eased exp ession o mi ogen-ac i a ed p o ein 4 kinase 4 (MAP4K4), which is known o inhibi PPAR γ ansc ip ional ac i i y, adipogenesis, and insulin-s imula ed glucose anspo [ 89 , 90 ]. In line wi h his, MAP4K4 inhibi s adipose lipogenesis ia he supp ession o S ebp-1 [ 91 ], and dele ion o his kinase inc eases insulin sensi i i y in adipose issue [ 92 ]. P olac in egula o y elemen -binding (PREB) is a nega i e egula o o adiponec in gene exp ession in adipocy es [93]. On he o he hand, EXOC7 migh pa icipa e in Glu 4 a icking [ 94 , 95 ], and SLC39A14 is a zinc anspo e , which is apidly induced in he ea ly s ages o di e en ia ion, sugges - ing a possible ole in adipogenesis [ 96 ]. In ac , deple ion o SLC39A14 caused hype ophy and in lamma ion o adipocy es [97]. These da a indica ed ha he combined pa ial knockdown o he MPST and CBS genes dis up s he pa e n o p o ein pe sul ida ion in human adipocy es, sugges ing a possible link be ween his dis up ion and adipocy e dys unc ion, which is cha ac e ized by dec eased adipogenesis bu inc eased in lamma ion. This dis up ion included a signi ican dec ease in pe sul ida ion in key p o eins o adipocy e unc ion (RAB4A, IDH2, NPC1, ACO1, LRP1, AHNAK, ARPC3, ADH1B, ALDH1A1, LGALS1, PHB, ANXA1, ANXA2, ACSL1, PLIN3, PLIN4, FABP4, HNRNPA1), bu also in p o eins ha nega i ely impac adipocy es and adipogenesis (ITGA5, COL6A3, ELAVL1). In addi ion, he combined pa ial knockdown o MPST and CBS genes in adipocy es esul ed in inc eased pe sul ida ion in ele an p oin lamma o y ansc ip ion ac o s (RELA, STAT1, STAT3) and an i-adipogenic p o eins (IFI16, MAP4K4, PREB), bu also in some p o eins ha migh exe posi i e e ec s on adipogenesis, such as SLC39A14 and EXOC7. Pe sul ida ion inc eases he eac i i y o a ge p o eins, modula ing hei biological ac i i ies [ 10 ]. Howe e , i is impo an o no e ha modula ion in pe sul ida ion-induced p o ein ac i i y can igge ac i a ion o inhibi ion o he biological unc ion o he pe sul ida ed p o ein [ 98 ]. O he consequences o CBS and MPST gene knockdown, such as dec eased H 2 S biosyn hesis [ 8 , 18 ] o inc eased homocys eine le els [ 99 ], could also explain he nega i e impac on adipocy e unc ion. Addi ional expe imen s using speci ic chemical inhibi o s o H 2 S biosyn hesis and he adminis a ion o H 2 S dono molecules o CBS/MPST gene knockdown cells should be pe o med o u he in es iga e he possible ole o H2S in his model. Selenium-binding p o ein 1 (SELENBP1), a ou h enzyme ha pa icipa es in H 2 S biosyn hesis, inc eased du ing di e en ia ion o 3T3L1 adipocy e in associa ion wi h lipoge- nesis and lipid accumula ion, and i has been ecen ly desc ibed as a ma ke o di e en ia ed adipocy es [ 100 ]. SELENBP1 gene knockdown had a nega i e impac on adipocy e unc ion in pa allel o he dec eased exp ession o he CBS, CTH, and MPST enzymes [ 101 ]. The con- ibu ion o o he enzymes, such as SELENBP1 [ 100 , 101 ], o non-enzyma ic pa hways [ 102 ] in H2S p oduc ion canno be excluded in he cu en s udy. 4. Conclusions In summa y, hese indings indica e ha he combined pa ial knockdown o CBS and MPST (bu no CTH) in adipocy es impai s he exp ession o genes ela ed wi h he main enance o adipocy e unc ion and p omo es in lamma ion, possibly by dis up ing he pa e n o p o ein pe sul ida ion in hese cells. The cu en s udy poin s o he ac ha