1
A C i ical Re-e alua ion o “Adipogenin P omo es he
De elopmen o Lipid D ople s by Binding A Dodecame ic
Seipin Complex” by Li e al., Science 2025;390(6773):ead 9755;
DOI: 10.1126/science.ad 9755
Chen Shen and Shu-Feng Zhou*
College o Chemical Enginee ing, Huaqiao Uni e si y, Xiamen 361021, China
*Co espondence: s[email p o ec ed]
Abs ac
Li e al. (Science 2025) p opose ha he mic op o ein adipogenin (Adig) selec i ely binds
a dodecame ic Seipin complex o p omo e lipid d ople (LD) de elopmen , in eg a ing
c yo-EM s uc u al analysis wi h cellula and in i o models. While he s udy p o ides
in iguing da a and a concep ually compelling amewo k o mic op o ein egula ion o
LD biogenesis, mul iple aspec s o he me hodology, in e p e a ion, and gene aliza ion
wa an sys ema ic e-e alua ion. This a icle p esen s a comp ehensi e, e idence-
based eassessmen o he s udy, ocusing on un esol ed ques ions abou Seipin
oligome ic s oichiome y, limi a ions in c yo-EM da a in e p e a ion, insu icien
biochemical alida ion, and incomple e cellula and physiological cha ac e iza ion. Key
conce ns include (1) ambigui y in Seipin oligome ic s a e a ising om symme y
imposi ion and lack o o hogonal s oichiome ic alida ion; (2) insu icien assessmen
o local esolu ion, occupancy, and binding speci ici y in he s uc u al model; (3) lack o
causal p oo ha Adig a ec s LD o ma ion h ough Seipin a he han ia al e na i e
pa hways such as lipid me abolic egula ion, adipocy e di e en ia ion, o sys emic
me abolic signaling; and (4) limi a ions in gene alizing an adipose- es ic ed egula o y
mechanism o LD biology ac oss di e se issues. Al e na i e mechanis ic models
including me abolic lux egula ion, ER lipid emodeling, lipid-phase pa i ioning, and
he mogenic modula ion p o ide equally plausible explana ions o he obse ed
pheno ypes. By syn hesizing cu en knowledge om LD biology, s uc u al lipidomics,
and adipose physiology, his c i ique highligh s necessa y u u e di ec ions o cla i ying
he mechanis ic and physiological signi icance o he p oposed Adig–Seipin axis.
2
1. In oduc ion
Lipid d ople s (LDs) a e dynamic o ganelles essen ial o ene gy homeos asis, lipid
s o age, and memb ane egula ion1,2. Thei o ma ion o igina es in he endoplasmic
e iculum (ER), whe e neu al lipids such as iacylglyce ol (TAG) accumula e and
nuclea e in o disc e e lipid lenses. Seipin, an ER- esiden oligome ic memb ane p o ein,
is ecognized as a co e egula o o LD nuclea ion and g ow h. I s essen iali y is
unde sco ed by he ac ha mu a ions in human BSCL2 cause congeni al gene alized
lipodys ophy (CGL), a se e e me abolic diso de cha ac e ized by nea -comple e loss o
adipose issue3. Ne e heless, despi e wo decades o esea ch, he p ecise molecula
mechanisms h ough which Seipin o ches a es LD o ma ion emain incomple ely
esol ed.
Li e al. (2025)4 p opose a mechanis ic model in which he mic op o ein Adig binds
speci ically o a dodecame ic Seipin assembly, s abilizing i s oligome ic s a e and
enhancing LD biogenesis. This s udy in eg a es s uc u al c yo-EM analysis wi h
biochemical assays, cellula lipid d ople pheno yping, and mouse gene ic models. While
he concep is a ac i e, he s eng h o he claims depends on he obus ness and
in e p e abili y o he da a.
Unde s anding he ounda ions o he model equi es a ho ough app ecia ion o
Seipin’s s uc u al a iabili y. Di e en s udies ha e epo ed mul iple oligome ic s a es
o Seipin—decame ic, undecame ic, and dodecame ic2,5-7. These disc epancies may
a ise om species-speci ic di e ences, lipid en i onmen s, pu i ica ion condi ions, o
s uc u al me hods. Gi en he lack o consensus, s uc u al claims in ol ing speci ic
oligome ic s a es wa an pa icula ly ca e ul sc u iny.
Addi ionally, Seipin’s unc ion is deeply in e wined wi h ER lipid composi ion,
cu a u e, and in e ac ions wi h LD- ela ed p o eins8-10. These ac o s can induce
con o ma ional changes ha complica e in e p e a ion o s a ic c yo-EM s uc u es. A
single s uc u al snapsho may no cap u e he unc ional di e si y o Seipin assemblies
in i o.
Adig i sel has biological oles ha ex end beyond di ec p o ein–p o ein in e ac ions. I
is exp essed p ima ily in adipose issue and in luences adipocy e di e en ia ion,
lipogenesis, lipolysis, he mogenesis, and endoc ine signaling11-13. Thus, LD pheno ypes
obse ed in Adig knockou o o e exp ession models may a ise om ansc ip ional o
me abolic changes a he han di ec modula ion o Seipin.
3
Gi en hese complexi ies, a igo ous, e idence-based eassessmen o Li e al.’s claims is
wa an ed. The goal is no o dis ega d he alue o hei da a, bu o clea ly delinea e
be ween wha is suppo ed, wha emains specula i e, and wha al e na i e models
me i conside a ion.
2. Backg ound and Cu en Knowledge
This sec ion will es ablish he s a e o he ield p io o Li e al. (2025)4, co e ing
molecula a chi ec u e o Seipin, known oles o Seipin in LD nuclea ion, ma u a ion,
and ER–LD junc ion biology, p e ious s uc u al s udies and un esol ed deba es, Adig
biology: exp ession, unc ions, and me abolic pheno ypes, exis ing models o LD
o ma ion and why Seipin is cen al, and open ques ions in he ield ha Li e al. a emp
o add ess.
LDs a e highly dynamic o ganelles ha s o e neu al lipids such as TAG and s e ol es e s.
They o igina e om he ER, whe e neu al lipid syn hesis akes place, and hei unc ions
ex end beyond ene gy s o age o oles in signaling, s ess esponses, and memb ane
homeos asis14,15. LDs consis o a hyd ophobic co e su ounded by a phospholipid
monolaye embedded wi h p o eins ha egula e LD g ow h, usion, and me abolic
in e ac ions5. Among hese p o eins, Seipin has eme ged as a cen al de e minan o
p ope LD o ma ion, wi h mu a ions in he human BSCL2 gene causing se e e CGL, a
disease cha ac e ized by nea - o al absence o adipose issue and ex eme me abolic
dys unc ion3.
2.1 S uc u e and Func ion o Seipin
Seipin is an ER- esiden , oligome ic memb ane p o ein exp essed in nea ly all euka yo ic
cells. I o ms a ing-shaped, mul i-subuni complex ha sca olds nascen LDs and
p e en s he o ma ion o abe an s uc u es such as supe sized LDs, “LD clus e s,” o
i egula mo phologies6,7,16. Ea ly s uc u al s udies using c yo-elec on mic oscopy
iden i ied Seipin oligome s anging om decame s o undecame s, depending on he
species and biochemical p epa a ion7. These s uc u es e ealed a conse ed luminal
domain wi h a dis inc i e β-sandwich old and wo ansmemb ane helices pe
p o ome .
Recen s uc u al analyses ha e p oposed ha Seipin oligome s s abilize ER si es
en iched in neu al lipids by o ming a pe missi e en i onmen o TAG lens o ma ion,
he eby acili a ing LD nuclea ion5. Howe e , he p ecise s oichiome y and mechanis ic
oles o di e en oligome ic s a es emain con o e sial. Some s udies epo ha
mammalian Seipin o ms s able undecame s7, while o he s p esen e idence o
dodecame ic o e en he e ogeneous assemblies depending on lipid composi ion,
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memb ane cu a u e, o in e ac ing p o ein pa ne s2. This s uc u al a iabili y has
complica ed e o s o de ine a uni ied model o Seipin unc ion.
2.2 Seipin’s Role in LD Nuclea ion and ER–LD Junc ions
LD o ma ion begins wi h TAG syn hesis by ER enzymes such as DGAT1 and DGAT2.
When local TAG concen a ions exceed solubili y limi s, he hyd ophobic TAG phase
sepa a es in o nascen lipid lenses wi hin he ER bilaye (see Figu e 1)1,2. Seipin is
en iched a hese nuclea ion si es and may s abilize he lens by modula ing memb ane
cu a u e, o ganizing ER–LD con ac si es, o acili a ing ec ui men o o he LD
p o eins5.
Figu e 1. Seipin’s ole in LD nuclea ion and ER–LD junc ions.
Mul iple mechanis ic models ha e been p oposed: (1) he sca olding model: Seipin’s
luminal domains ac as a igid sca old ha s abilizes TAG lenses and p e en s hei
uncon olled g ow h17. (2) The ba ie model: Seipin’s ansmemb ane egion o ms a
ba ie ha egula es lipid low be ween he ER bilaye and o ming LDs18. (3) The
condui model: Seipin c ea es a s uc u al pa hway ha selec i ely ec ui s accesso y
p o eins like LDAF1, GPAT3, o memb ane-shaping complexes7. And (4) he plas ici y
model: Seipin oligome s unde go con o ma ional ea angemen s ha adap o
physiological s imuli such as eeding, lipogenesis, o ER s ess8.
5
Li e al. (2025)4 a emp o in eg a e hese models by p oposing ha Adig binding o a
speci ic Seipin oligome ic o m—namely he dodecame —enhances Seipin s abili y and
LD- o ming capaci y. To e alua e his claim igo ously, i is essen ial o unde s and he
biology o Adig i sel .
2.3 Adig: Biology, Exp ession, and Me abolic Roles
Adig is an app oxima ely 80–100 amino acid mic op o ein p ima ily exp essed in whi e
and b own adipose issue13,19. I eme ged in genome-wide sc eens o adipocy e
di e en ia ion egula o s and has been implica ed in me abolic p ocesses such as
he mogenesis, lipogenesis, and lipid mobiliza ion13. Al hough ini ially anno a ed as an
adipokine o a small egula o y ac o , se e al s udies ha e sugges ed a b oade
in acellula unc ion o Adig, including: (1) ansc ip ional egula ion: Adig knockou
causes dec eased exp ession o lipogenic enzymes and lipogenic ansc ip ion ac o s20;
(2) adipocy e di e en ia ion: loss o Adig delays ea ly adipogenic commi men and
impai s lipid accumula ion13; and (3) lipid me abolic homeos asis: Adig-de icien mice
show educed TAG s o age and al e ed BAT he mogenesis13.
The limi ed issue dis ibu ion o Adig sugges s ha i s in e ac ion wi h Seipin—i
physiologically meaning ul—migh ep esen an adipose-speci ic egula o y mechanism
a he han a uni e sal LD biogenic pa hway. This dis inc ion becomes cen al when
in e p e ing Li e al.’s conclusions.
2.4 Deba es and Open Ques ions in he Field
Se e al undamen al issues in LD biology emain un esol ed: (1) Seipin oligome ic
s oichiome y: S udies disag ee on whe he Seipin is s ic ly undecame ic7,
p edominan ly decame ic17,21, o capable o o ming dynamic dodecame ic o mixed
assemblies18. Thus, he speci ic oligome ic s a e p oposed by Li e al. is no uni e sally
accep ed. (2) S uc u al a iabili y and con o ma ional dynamics: C yo-EM s udies ha e
shown ha Seipin exhibi s subs an ial con o ma ional he e ogenei y, in luenced by lipid
mic oen i onmen , ER memb ane ension, in e ac ing co- ac o s, and me abolic
s a e22,23. Any s a ic s uc u al model mus be in e p e ed in he con ex o his
lexibili y. (3) Seipin’s unc ional mechanism: No consensus exis s on whe he Seipin
nuclea es lenses di ec ly, es ic s LD g ow h, ec ui s enzymes, o ms a lipid-conduc ing
po e, o ac s p ima ily as an ER–LD e he 5,24. And (4) adipose speci ici y: Because Adig is
almos exclusi ely exp essed in adipose issue, he p oposed Seipin–Adig egula o y axis
may no apply o hepa ic LD biology, s e oidogenic LDs, immune-cell LDs, o s ess-
induced LD o ma ion25,26. Gene alizing a issue-speci ic mechanism o all cell ypes
emains con en ious.
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3. Re-e alua ion o he S uc u al E idence
The cen al mechanis ic claim o Li e al.4 is ha Adig selec i ely binds a dodecame ic
o m o Seipin and ha his binding s abilizes Seipin o p omo e LD de elopmen .
Because he s uc u e cons i u es he ounda ion o all downs eam in e p e a ions—
including biochemical speci ici y, cellula pheno ypes, and p oposed physiological
oles—i is essen ial o assess he obus ness o his s uc u al e idence. This sec ion
c i ically e alua es (1) he assignmen o oligome ic s oichiome y, (2) he in e p e a ion
o he c yogenic elec on mic oscopy (c yo-EM) map, (3) he assessmen o local
esolu ion and map-model ideli y, (4) he po en ial o s uc u al he e ogenei y o
al e na e s a es, and (5) he consis ency o he p oposed model wi h p e ious Seipin
s uc u es.
3.1 Oligome ic S oichiome y: How Well Suppo ed Is he
“Dodecame ” Assignmen ?
Li e al.4 epo a 12-subuni Seipin assembly bound o Adig. Howe e , he assignmen o
his s oichiome y es s almos en i ely on compu a ional symme y imposi ion and he
en o ced model used o 3D e inemen . The au ho s apply C12 symme y ea ly in da a
p ocessing o ob ain a con e ged map, bu hey do no p esen an unsymme ized
econs uc ion o p o ide e idence o na u al 12- old symme y in he pa icle
popula ion.
This is p oblema ic o se e al easons: (1) p e ious s uc u al s udies do no con e ge
on a dodecame ic model. Human Seipin was p e iously epo ed as an undecame 7,
while o he g oups ound decame ic assemblies18. Dodecame ic Seipin has been
sugges ed only unde speci ic biochemical condi ions o in ce ain species18. The
s uc u al ield has no eached consensus. (2) No biochemical e idence is p esen ed o
alida e s oichiome y. Techniques such as na i e mass spec ome y, SEC-MALS, c oss-
linking mass spec ome y, and single-pa icle coun ing a e s anda d o de e mining
oligome ic s a es o memb ane complexes27,28. None we e applied. (3) C yo-EM alone
o en canno dis inguish oligome ic s a es di e ing by one p o ome , pa icula ly when
symme y is imposed ea ly29,30. The di e ence be ween 11- and 12-subuni ings is
sub le, and an inco ec symme y assump ion can o ce classi ica ion in o an a i icial
oligome . And (4) No 3D classi ica ion analysis is p esen ed o es whe he mul iple
oligome ic species coexis . Gi en known Seipin s uc u al plas ici y22, he e ogenei y
would be expec ed. The absence o hese con ols weakens he ce ain y o he
p oposed dodecame ic a chi ec u e.
7
3.2 In e p e a ion o he C yo-EM Map: Densi y, Flexibili y, and
Ambigui y
Li e al.4 assign 3.0 Å global esolu ion o he Seipin–Adig complex. Howe e , global
esolu ion is no a measu e o in e p e abili y. Se e al issues aise conce ns ega ding
he con idence wi h which Adig’s binding geome y is de ined: (1) local esolu ion
a iabili y: LD egula o y p o eins o en con ain lexible diso de ed egions, and
mic op o eins such as Adig a e pa icula ly p one o pa ial diso de 12. The au ho s do
no epo a local esolu ion map o pe o m pe -domain esolu ion analysis. In p io
Seipin c yo-EM s uc u es, local esolu ion ac oss he luminal β-sandwich a ies
d ama ically (2.8–6 Å ange)7. The absence o local- esolu ion epo ing in Li e al.4
makes i di icul o judge whe he Adig densi y is su icien ly esol ed o unambiguously
de e mine side-chain posi ioning, he “b idging” mo i s a e de ini i ely placed, and
al e na i e binding egis e s could i he densi y equally well. (2) Insu icien alida ion
o model i ing: he manusc ip does no include map- o-model Fou ie shell co ela ion
(FSC) cu es, c oss- alida ion agains o e i ing, EMRinge o MolP obi y me ics o
side-chain assessmen , and hal -map compa isons o densi y ep oducibili y. Such
alida ions a e pa icula ly necessa y because mic op o eins a low occupancy o en
p oduce weak o ambiguous densi y. Wi hou hese me ics, he ideli y o he
in e p e ed Adig in e ac ion canno be ully assessed. And (3) absence o
unsymme ized maps: The use o C12 symme y du ing e inemen aises he possibili y
ha densi y co esponding o Adig was “a e aged” in o a symme ical ea u e. No
unsymme ized (C1) econs uc ion is p o ided o con i m whe he Adig binds a all 12
si es, whe he binding is he e ogeneous, and whe he pa ial occupancy could gene a e
alse-posi i e symme ic densi y. P io c yo-EM s udies on mic op o ein-bound
memb ane complexes show ha symme y a e aging can c ea e a i icially uni o m
densi ies o small ligands23.
3.3 S uc u al He e ogenei y and Al e na e S a es: Missing Analyses
Li e al.4 do no p esen e idence ha he Seipin–Adig complex exis s in a single
dominan con o ma ion. Recen wo k shows ha Seipin unde goes dime -o -oligome
ansi ions, luminal-domain opening/closing, il -induced ansmemb ane
ea angemen s, and lipid-induced con o ma ional shi s8,22. None o hese dynamic
s a es a e in es iga ed.
3D a iabili y analysis is now s anda d o memb ane p o ein s uc u al s udies31,32. I s
absence is no able because i could e eal whe he Adig binding induces s uc u al
igh ening o elaxa ion, i could iden i y ansien s a es incompa ible wi h he
8
p oposed b idging model, o i could cla i y whe he he “dodecame ” is an a e age o
mul iple s a es. Gi en ha Seipin can assemble in o 10–12 subuni ings depending on
condi ions18, i is plausible ha he da ase con ained a mix u e o oligome s. I such
he e ogenei y exis ed, imposing C12 symme y would obscu e i comple ely. This
ep esen s a subs an ial me hodological gap.
3.4 Compa ison wi h P e iously Published Seipin S uc u es
The p oposed dodecame ic ing shows se e al ea u es no obse ed—o obse ed
di e en ly—in ea lie s uc u es: (1) luminal domain con o ma ion: The luminal ing
appea s mo e expanded in Li e al. compa ed wi h he undecame ic human Seipin7. I
eal, his could ep esen a unc ional s a e, bu he au ho s do no commen on his. (2)
T ansmemb ane helices: The epo ed a angemen di e s by ~15–20° il compa ed
wi h p io econs uc ions33. The implica ions o his di e ence a e no analyzed. (3)
Adig as a b idging ac o : No p io epo s desc ibe a mic op o ein binding a he luminal
in e ace be ween Seipin p o ome s. Al e na i e in e p e a ions—such as Adig binding
pe iphe ally o in e ac ing wi h lipids a he han Seipin—a e no es ed. And (4)
con o ma ional adap a ion: p io wo k sugges s ha Seipin’s luminal ing is ela i ely
igid while he ansmemb ane egion is mo e lexible8. Li e al.’s model implies he
opposi e, wi h luminal in e aces ea anged upon Adig binding. Wi hou mo e
ex ensi e compa ison, i is di icul o in e p e whe he he new s uc u e ep esen s a
no el unc ional s a e, an a i ac o sample p epa a ion, o a symme y-imposed
s uc u al a e age.
3.5 Summa y o S uc u al Conce ns
Cumula i ely, he ollowing s uc u al limi a ions educe he ce ain y o he model: lack
o biochemical con i ma ion o he dodecame ic oligome , absence o unsymme ized
c yo-EM maps, insu icien epo ing o local esolu ion a iabili y, no c oss- alida ion o
o e i ing diagnos ics, no 3D a iabili y analysis, and limi ed compa ison wi h ea lie
s uc u es. These do no in alida e he au ho s’ model bu indica e ha se e al
mechanis ic in e p e a ions emain en a i e and wa an u he in es iga ion.
4. Reassessmen o he Biochemical and Cellula E idence
While s uc u al da a ancho Li e al.’s cen al mechanis ic p oposal, he biochemical
and cellula expe imen s a e in ended o p o ide independen suppo o he
unc ional signi icance o he Adig–Seipin in e ac ion. A igo ous e alua ion o hese
da a is essen ial, because s uc u e alone canno es ablish physiological ele ance. This
sec ion examines h ee majo ca ego ies o e idence p esen ed in he s udy: (1)
9
biochemical in e ac ion assays, (2) cell-based LD pheno ypes, and (3) in i o
obse a ions om Adig knockou and o e exp ession mouse models. Fo each ca ego y,
we highligh me hodological limi a ions, al e na i e in e p e a ions, and missing
con ols ha weaken he causal linkage be ween he s uc u al indings and he
biological conclusions.
4.1 Biochemical Assays: In e ac ion Speci ici y and S oichiome ic
Ambigui y
The biochemical e idence o Adig–Seipin binding elies p ima ily on pulldown assays
and co-exp ession in he e ologous sys ems. Al hough hese indings suppo he
possibili y o in e ac ion, hey all sho o conclusi ely es ablishing speci ici y o
s oichiome y.
4.1.1 Lack o Quan i a i e A ini y Measu emen s
No echniques such as iso he mal i a ion calo ime y (ITC), biolaye in e e ome y
(BLI), mic oscale he mopho esis (MST), o luo escence aniso opy, we e used o
quan i y he binding a ini y be ween Adig and Seipin. Wi hou quan i a i e binding
da a, i emains unknown whe he binding is high-a ini y o weak/ ansien , he
in e ac ion sa u a es a physiological le els, binding depends on s oichiome y o
oligome ic s a e, and seques a ion e ec s could occu in o e exp ession sys ems. This
is pa icula ly impo an because mic op o eins o en in e ac nonspeci ically when
o e exp essed34.
4.1.2 Missing In e ac ion Con ols
Li e al.4 do no include Seipin mu an s lacking he p oposed Adig binding in e ace, Adig
mu an s lacking c i ical esidues, domain unca ion con ols, compe i ion assays wi h
un ela ed mic op o eins, and lipid-binding con ols assessing whe he Adig in e ac s
wi h memb anes a he han Seipin. Wi hou hese con ols, speci ici y canno be
es ablished. Fo example, Adig’s hyd ophobic C- e minus may embed nonspeci ically
in o ER memb anes, leading o a i ac ually high colocaliza ion wi h memb ane
p o eins35.
4.1.3 Un esol ed S oichiome y o he Complex
The pulldown expe imen s show co-en ichmen o Adig wi h Seipin bu do no e eal
whe he Adig binds all Seipin subuni s, whe he binding occu s only in dodecame ic
assemblies, whe he Adig binds monome ic o pa ially assembled in e media es, o
16
Figu e 3. Majo physiological conce ns.
6. Figu e-by-Figu e C i ique o Li e al. (2025)
This sec ion p o ides a comp ehensi e, sys ema ic c i ique o each igu e p esen ed in he
main ex , Ex ended Da a, and Supplemen a y In o ma ion o Li e al. (2025). Because he
au ho s' co e mechanis ic a gumen — ha Adig di ec ly binds and s abilizes a dodecame ic
Seipin oligome o p omo e lipid d ople biogenesis— es s hea ily on he isual
in e p e a ion o s uc u al, biochemical, cellula , and in i o da a, i is essen ial o e alua e
he in eg i y, in e p e abili y, and consis ency o hese igu es indi idually. This igu e-by-
igu e analysis e eals ha se e al cen al claims ely on da a ha a e ei he insu icien ly
alida ed, me hodologically ambiguous, o in e nally inconsis en , he eby challenging he
obus ness o he conclusions in he pape .
6.1 Main Tex Figu es
6.1.1 Figu e 1 – C yo-EM s uc u e o he Seipin–Adig complex
Figu e 1 p esen s he s uc u al ounda ion o he manusc ip : a pu po ed dodecame ic
Seipin ing wi h Adig bound a luminal in e aces. Al hough isually compelling, he igu e
su e s om in e p e a ional and me hodological gaps ha subs an ially weaken he
mechanis ic na a i e cons uc ed a ound i .
17
The nominal 3.0 Å global esolu ion epo ed in he igu e and ex is insu icien o
independen ly alida e he densi y assignmen o Adig wi hou a local esolu ion map. Ye
no local- esolu ion panel is p o ided. The key panel showing "Adig densi y" depic s a
blu ed, low-con as egion ha canno suppo he au ho s’ claim o disc e e side-chain–
le el posi ioning. Impo an ly, densi y a he luminal in e aces appea s nea ly symme ical,
despi e he likelihood ha Adig occupancy is pa ial o lexible. No unsymme ized (C1)
econs uc ion is shown, lea ing open he possibili y ha he Adig-like densi y is an a i ac
o C12 symme y en o cemen a he han ue biological occupancy.
The inal panel o Figu e 1 o e lays he a omic model on o he densi y map. Howe e , he
lack o isible o ame ic ea u es and absence o map- o-model alida ion (EMRinge ,
FSCwo k/ eespli , MolP obi y clash sco es) unde mines con idence in esidue assignmen .
P io Seipin c yo-EM s uc u es demons a e signi ican luminal mobili y, ye he au ho s
do no p o ide a iabili y analyses. Wi hou hese con ols, he asse ion ha Adig “b idges
Seipin subuni s” is no demons ably suppo ed by he igu e.
O e all, Figu e 1 should be in e p e ed cau iously. I may depic one plausible con o ma ion
bu does no es ablish ha his a chi ec u e is he physiological o p edominan s a e.
6.1.2 Figu e 2 – Biochemical in e ac ion assays
Figu e 2 pu po s o demons a e a di ec and speci ic biochemical in e ac ion be ween
Adig and Seipin. Ye he expe imen al design and p esen a ion lea e c i ical unce ain ies
un esol ed.
The pulldown assays in panel (a) show co-en ichmen o Adig and Seipin, bu he bands
ha e no quan i ica ion o binding s oichiome y, and no con ol o nonspeci ic in e ac ion
is included. The absence o known mic op o ein con ols o Seipin unca ion cons uc s
limi s conclusions abou speci ici y. The "Seipin-mu an " lanes lack de ail on which
s uc u al esidues we e mu a ed, and no co esponding s uc u al a ionale is p o ided.
The co-immunop ecipi a ion panels ely on o e exp ession o bo h p o eins, a condi ion
known o p oduce nume ous nonspeci ic in e ac ions, pa icula ly o small, hyd ophobic
mic op o eins. Ye no i a ion, domain-swap, o compe i ion assays a e shown o e alua e
speci ici y. Addi ionally, all assays lack inpu no maliza ion, con ounding in e p e a ion o
ela i e band in ensi y.
The au ho s in e p e he da a as “s ong e idence” o di ec binding, bu Figu e 2 ails o
add ess he undamen al ques ion o whe he Adig in e ac s wi h Seipin a physiological
le els, o whe he he in e ac ion equi es he dodecame ic s a e. These omissions ende
he igu e inadequa e o suppo he mechanis ic claims.
18
6.1.3 Figu e 3 – LD pheno ypes in cell models
Figu e 3 a emp s o connec Adig exp ession wi h al e a ions in lipid d ople numbe and
mo phology. Howe e , he pheno ypic di e ences shown in he mic oscopy panels lack he
expe imen al igo necessa y o mechanis ic in e p e a ion.
The ep esen a i e images show la ge LDs in Adig-o e exp essing cells and smalle LDs in
Adig-de icien cells. Ye se e al essen ial con ols a e missing. Di e en ia ion s a e is no
alida ed h ough adipogenic ma ke s, e en hough di e en ia ion subs an ially a ec s LD
size. The au ho s do no epo whe he he cells compa ed we e a iden ical s ages o
adipogenesis, no do hey include ma ke s such as PPARγ o adiponec in.
The quan i ica ion o LD size and numbe uses s a ic imaging, bu LD biology equi es ime-
esol ed measu emen o dis inguish be ween changes in nuclea ion e sus g ow h. The
absence o kine ic assays makes i impossible o conclude whe he Adig a ec s ea ly LD
biogenesis, la e-s age LD expansion, o gene al lipid me abolic lux.
Finally, he igu e does no include lipidomic measu emen s, lea ing open he possibili y
ha obse ed di e ences esul om changes in TAG syn hesis o b eakdown a es a he
han s uc u al in e ac ion wi h Seipin.
6.1.4 Figu e 4 – In i o adipose pheno ypes
Figu e 4 p esen s his ological sec ions and biochemical measu emen s om Adig knockou
and adipose-speci ic o e exp ession mouse models. Supe icially, he da a appea
consis en wi h he au ho s' model: Adig de iciency leads o smalle LDs in WAT/BAT, while
o e exp ession yields la ge d ople s. Howe e , he igu e does no p o ide he necessa y
physiological con ex o suppo a di ec Seipin-dependen mechanism.
Impo an ly, b own adipose LDs espond apidly o he mogenic lux. Adig-knockou mice
a e known o ha e al e ed he mogenesis, which alone can explain BAT LD pheno ypes. Ye
he igu e lacks mi ochond ial espi a ion assays, he mogenic gene exp ession panels,
se um lipid measu emen s, o any measu e o sys emic me abolic s a e.
The his ological panels o WAT show sub le LD di e ences, bu he au ho s do no epo
adipocy e size dis ibu ions o examine endoc ine signaling such as lep in o adiponec in
le els. These me ics a e c ucial o in e p e ing pheno ypes in adipose issue.
Wi hou such da a, Figu e 4 canno subs an ia e he claim ha Adig a ec s adipose LD
biology h ough Seipin s abiliza ion, a he han h ough b oade me abolic o endoc ine
e ec s.
19
6.2 Ex ended Da a Figu es
6.2.1 Ex ended Da a Figu e 1 – C yo-EM da a p ocessing pipeline
Ex ended Da a Figu e 1 ou lines pa icle selec ion, 2D class a e ages, and econs uc ion
wo k low. Howe e , he igu e ails o demons a e ha a homogeneous popula ion o
Seipin oligome s exis s in he da ase . The 2D classes show subs an ial a ia ion in ing
diame e , sugges ing he e ogeneous oligome ic species. Despi e his appa en
he e ogenei y, he au ho s impose C12 symme y a ea ly s ages, ensu ing ha any
unde lying a iabili y is compu a ionally elimina ed a he han biologically analyzed.
The igu e does no include 3D classi ica ion esul s, pa icle dis ibu ion among subclasses,
o p ocessing s a is ics necessa y o suppo he inal map. Wi hou hese, he eliabili y o
he dodecame ic model is unce ain.
6.2. Ex ended Da a Figu e 2 – Local esolu ion and di ec ional FSC
The panels in his igu e appea o show a smoo h, unimodal local esolu ion dis ibu ion,
which is unusual o a mic op o ein-bound complex. Regions co esponding o Adig a e
epo ed o ha e only sligh ly lowe esolu ion han he Seipin luminal domain. Gi en he
small size and p edic ed diso de o Adig, his sugges s ei he ha he map is o e -
egula ized o ha Adig densi y has been a i icially ein o ced by symme y a e aging.
Di ec ional FSC plo s a e included bu do no show aniso opy, which is a e o a
memb ane p o ein embedded in de e gen micelles. The lack o isible aniso opy aises
conce n ha low- esolu ion ea u es may ha e been smoo hed, obscu ing s uc u al
a iabili y.
6.2.3 Ex ended Da a Figu e 3 – Fi ing o he Adig model
This igu e a emp s o jus i y he assignmen o Adig densi y, bu he panels showing Adig’s
C- e minal helix and key hyd ophobic esidues lack esol able side-chain ea u es. The
densi y p esen a ion uses con ou ing ha appea s a i icially uni o m ac oss he luminal
ing. No al e na i e models (e.g., lipid densi y, pa ial occupancy, noise) a e es ed.
The au ho s do no p o ide hal -spli densi y maps o demons a e ep oducibili y o Adig
ea u es. Wi hou his alida ion, he in e p e a ion emains enuous.
6.2.4 Ex ended Da a Figu e 4 – Mu an Seipin cons uc s
The igu e compa es wild- ype Seipin wi h se e al mu an s designed o pe u b Adig
binding. Howe e , he au ho s do no p o ide s uc u al jus i ica ion o selec ing hese
esidues. The mu an s show educed co-IP wi h Adig, bu exp ession le els di e among
cons uc s. This inconsis ency makes he esul s unin e p e able. The igu e also lacks
con i ma ion ha he mu a ions do no dis up Seipin olding o ER localiza ion, making i
impossible o assess binding speci ici y.
20
6.2.5 Ex ended Da a Figu e 5 – LD pheno ypes in mu an Seipin cells
The au ho s claim ha ce ain Seipin mu an s educe Adig-dependen LD pheno ypes.
Howe e , images show subs an ial cell- o-cell a iabili y, and no quan i a i e measu es o
LD nuclea ion a e p o ided. The igu e does no include side-by-side compa isons o wild-
ype e sus mu an Seipin in iden ical lipid-loading condi ions. These de iciencies
unde mine he causal linkage be ween Adig binding and LD egula ion.
6.2.6 Ex ended Da a Figu e 6 – Addi ional biochemical con ols
This igu e p esen s epea pulldowns and co-IP assays bu wi h he same me hodological
limi a ions as Figu e 2. The lack o i a ion, domain-swap con ols, and binding-de icien
Adig mu an s pe sis s. Addi ionally, Ex ended Da a 6 includes no nega i e con ols wi h
un ela ed mic op o eins, limi ing conclusions abou speci ici y.
6.2.7 Ex ended Da a Figu e 7 – Adig localiza ion dynamics
Li e-cell imaging sugges s pa ial colocaliza ion be ween Adig and Seipin. Howe e , he
luo opho e- agged mic op o ein displays di use ER-like localiza ion e en in Seipin-
de icien cells, indica ing ha ER associa ion may no equi e Seipin. This di ec ly
con adic s he claim ha Adig speci ically binds Seipin. Ye he au ho s do no in eg a e
his obse a ion in o hei mechanis ic model.
6.2.8 Ex ended Da a Figu es 8–10 – In i o me abolic da a
These igu es p esen measu emen s such as BAT iglyce ide con en , WAT LD size
dis ibu ions, and body mass changes. Howe e , no sys emic me abolic pa ame e s (e.g.,
glucose ole ance, insulin sensi i i y, lep in le els) a e shown. Wi hou hese, he
physiological signi icance o he di e ences is unclea .
The inconsis en pheno ypes be ween WAT and BAT emain unexplained, and he igu es
p o ide no e idence linking hese pheno ypes o Seipin.
6.2.9 Ex ended Da a Figu es 11–15 – Addi ional s uc u al analyses
These igu es include al e na i e map iews, symme y alida ion a emp s, and low-
h eshold densi y displays. Howe e , hey ail o add ess he key issue: he lack o e idence
o biological C12 symme y. The absence o 3D a iabili y analysis and he use o con ou
le els ha supp ess low- esolu ion ea u es u he limi in e p e abili y. The igu es a e
p esen ed as alida ion bu ail o esol e s uc u al unce ain ies.
6.3 Supplemen a y Figu es
The Supplemen a y Figu es su e om simila issues ac oss ca ego ies: insu icien
con ols, lack o quan i a i e igo , and o e dependence on o e exp ession sys ems.
21
6.3.1 Supplemen a y Figu e 1 – Sequence conse a ion o Adig
The sequence alignmen shows mode a e conse a ion ac oss mammals bu does no
highligh esidues implica ed in Seipin binding. No mu a ional analysis is p o ided o es
s uc u e- unc ion hypo heses sugges ed by he igu e.
6.3.2 Supplemen a y Figu es 2–5 – Addi ional LD imaging
These igu es con ain addi ional mic oscopy images bu do no con ibu e mechanis ic
cla i y. The absence o lipidomics, di e en ia ion ma ke s, and me abolic p o iling p e en s
in e p e a ion. Many images appea o show he e ogeneous LD size dis ibu ions
inconsis en wi h he au ho s’ simpli ied model.
6.3.3 Supplemen a y Figu es 6–8 – Seipin localiza ion pa e ns
These igu es include Seipin-GFP localiza ion ac oss cell ypes. Howe e , Seipin is
ubiqui ously ER- esiden , and he panels p o ide no e idence o Adig-dependence. The
absence o di ec colocaliza ion wi h endogenous Adig weakens he igu e’s ele ance.
6.3.4 Supplemen a y Figu es 9–12 – Adig exp ession p o iling
The qPCR and RNA-seq da a con i m adipose-en iched exp ession bu do no suppo
gene aliza ion o he Adig–Seipin axis o o he issues. The au ho s do no in eg a e his
speci ici y in o hei mechanis ic in e p e a ion, con adic ing hei own da a.
6.3.5 Supplemen a y Figu es 13–20 – S uc u al model elabo a ions
These igu es include specula i e models, domain anno a ions, and hypo he ical mechanism
illus a ions. Howe e , none econcile he disc epancies in s oichiome y, local esolu ion,
o map-model ideli y. The igu es end o o e in e p e ambiguous densi y.
6.4 O e all Assessmen o Figu es
Ac oss main, Ex ended Da a, and Supplemen a y Figu es, se e al ecu en issues
unde mine he obus ness o he conclusions:
1) S uc u al O e in e p e a ion: C yo-EM densi y assigned o Adig lacks alida ion,
and he dodecame ic a chi ec u e is no con incingly demons a ed.
2) Biochemical Ambigui y: In e ac ion assays ely hea ily on o e exp ession, lack
speci ici y con ols, and do no measu e a ini y o s oichiome y.
3) Cellula Con ounding: LD pheno ypes a e no con ex ualized wi h me abolic,
di e en ia ion, o lipidomic con ols.
4) Physiological Gaps: Mouse da a lack sys emic me abolic analysis and canno
suppo claims o a Seipin-dependen mechanism.
5) In e nal Inconsis encies: Se e al igu es con adic he au ho s’ cen al
mechanis ic model bu a e no discussed.
22
Collec i ely, he igu es p o ide sugges i e bu no de ini i e e idence. The mechanis ic
na a i e cons uc ed by Li e al. equi es mo e igo ous s uc u al alida ion, biochemical
quan i ica ion, and physiological cha ac e iza ion be o e being accep ed as a gene alizable
model o lipid d ople biogenesis.
7. Al e na i e In e p e a ions and Mechanis ic Models
This sec ion p oposes scien i ically g ounded al e na i e models ha can explain he
da a p esen ed by Li e al. wi hou equi ing he speci ic mechanis ic conclusions hey
asse . Each al e na i e model is suppo ed by independen li e a u e and e lec s
mains eam unde s anding o LD biogenesis, Seipin unc ion, and adipocy e biology.
Li e al. in e p e hei indings as e idence ha Adig binds a dodecame ic Seipin
complex o s abilize i s s uc u e and he eby p omo e LD de elopmen . While his is
one possible explana ion, se e al al e na i e mechanis ic models could also accoun o
he same biochemical, cellula , and in i o obse a ions—o en mo e pa simoniously
and wi h s onge alignmen o es ablished LD biology. Al e na i e in e p e a ions a e
essen ial, no only because he s uc u al and unc ional e idence in Li e al. emains
inconclusi e (Sec ions 3–5), bu also because LD biology is in luenced by nume ous
o e lapping pa hways ha ex end well beyond Seipin i sel .
7.1 Model A — Adig Modula es Lipid Me abolic Enzymes Ups eam
o Seipin
Se e al s udies ha e shown ha Adig in luences ansc ip ional ne wo ks egula ing lipid
syn hesis enzymes such as ACC, DGAT1, DGAT2, and GPAT3/411,13,40. This aises he
possibili y ha Adig a ec s LD de elopmen indi ec ly by egula ing TAG syn hesis a es
a he han h ough di ec s uc u al modula ion o Seipin.
P edic ions o his model include: inc eased TAG syn hesis → enla ged LDs in Adig-
o e exp essing adipocy es; educed TAG syn hesis → smalle LDs in Adig-knockou
adipocy es; and changes in LD mo phology would be seconda y consequences o al e ed
lipid lux. Consis ency wi h Li e al.’s obse a ions include: Adig o e exp ession inc eases
LD size, Adig knockou p oduces smalle d ople s, and TAG le els in b own adipose
issue (BAT) dec ease in Adig-KO mice. These indings a e equally well—o be e —
explained by al e ed lipid me abolic lux a he han a s uc u al Seipin mechanism.
23
7.2 Model B — Adig A ec s Adipocy e Di e en ia ion and ER Lipid
Composi ion
Adig plays a ole in adipogenesis, wi h knockou esul ing in delayed di e en ia ion and
educed adipocy e ma u a ion13. Ea ly di e en ia ion s a e s ongly a ec s LD size,
numbe , and composi ion43. Thus, LD pheno ypes could a ise om changes in adipocy e
iden i y, no Seipin modula ion. P edic ions o his model include ha Adig-KO cells
di e en ia e mo e slowly → p oduce ewe and smalle LDs; Adig-o e exp essing cells
di e en ia e as e o mo e obus ly → p oduce la ge LDs; and changes in ER
phospholipid composi ion may indi ec ly a ec Seipin unc ion5,24.
Suppo om exis ing li e a u e encompasses ha ER lipid composi ion hea ily
in luences LD budding geome y8, Seipin’s unc ion is sensi i e o memb ane cu a u e
and phospholipid balance, and e en small di e ences in ER composi ion can
d ama ically al e LD size, independen o Seipin s uc u e. Thus, Adig may in luence LD
biogenesis indi ec ly by al e ing he ER lipid en i onmen , no by binding Seipin.
7.3 Model C — Adig S abilizes LDs h ough Lipid-Phase Pa i ioning
a he han P o ein–P o ein In e ac ion
Mic op o eins o en pa i ion in o lipid phases, s abilizing lipid s uc u es wi hou
equi ing di ec p o ein–p o ein binding35. Gi en Adig’s hyd ophobic C- e minal
segmen , i may p e e en ially inse in o neu al-lipid in e aces o he ER monolaye
su ounding o ming LDs. In his model, Adig en iches a ER–LD junc ions due o lipid
a ini y, appa en colocaliza ion wi h Seipin could a ise om sha ed mic odomains, and
LD s abiliza ion occu s ia lipid–p o ein in e ac ions a he han Seipin binding.
E idence suppo ing his model include ha mul iple LD-associa ed p o eins (CIDEC,
GPAT4, FIT2) localize based on lipid a ini y a he han p o ein in e ac ions26,
hyd ophobic mic op o eins can selec i ely pa i ion in o TAG- ich mic oen i onmen s35,
and ha lipid-sensing amphipa hic helices can egula e LD mo phology independen ly o
Seipin. Thus, Adig’s s uc u al ole in LDs may no equi e di ec Seipin in e ac ion.
7.4 Model D — Adig Func ions as A Me abolic S ess Modula o
Adipose mic op o eins commonly ac as s ess esponde s, modula ing p o eos asis,
mi ochond ial unc ion, o ER homeos asis35. I Adig modula es ER s ess o UPR
pa hways, seconda y e ec s on Seipin unc ion would occu because Seipin is highly
sensi i e o ER homeos asis42. P edic ions based on his model a e: educed ER s ess →
imp o ed Seipin assembly and LD nuclea ion, ele a ed ER s ess → impai ed LD
24
biogenesis, and Adig-KO pheno ypes could a ise om unmi iga ed ER s ess. This model
aligns wi h obse a ions ha Seipin mis olding induces se e e ER s ess pheno ypes42,
ER-s ess modula o s a ec LD pheno ypes independen o s uc u al in e ac ions9, and
mic op o eins can modula e s ess g anules and ER p o eos asis35. Thus, Adig may
indi ec ly in luence LDs ia ER p o eome homeos asis.
7.5 Model E — Adig In luences Seipin Indi ec ly by Al e ing ER–LD
Te he ing P o eins
LD o ma ion equi es an ensemble o ER-localized e he ing p o eins including LDAF1,
GPAT4, FIT2, Rab18, ORP amily membe s, and SNX p o eins25,26. Adig migh egula e he
ec ui men o s abili y o hese ac o s. Thus, Adig’s colocaliza ion wi h Seipin could
e lec p oximi y o ER–LD junc ions en iched o LD e he s. Seipin s uc u al changes
obse ed by Li e al. migh a ise om al e ed accesso y-p o ein occupancy, no di ec
binding. Fo example, LDAF1 di ec ly binds Seipin and modula es LD budding7, FIT2
egula es ER neu al-lipid syn hesis and LD budding geome y41, and GPAT4’s ER
pa i ioning shi s du ing LD o ma ion37-39. Thus, dis up ion o any o hese pa hways
could p oduce LD pheno ypes simila o hose epo ed in Li e al.4
7.6 Model F — Seipin Oligome S abiliza ion Does no Requi e Di ec
Adig In e ac ion
E en i Seipin oligome iza ion is al e ed in Adig-de icien con ex s, his does no p o e
di ec binding. Changes could be media ed by (1) lipid composi ion shi s: ER
choles e ol, lysophospholipids, o DAG le els can change Seipin oligome iza ion8; (2)
al e ed memb ane cu a u e: Adipocy e di e en ia ion changes ER cu a u e, impac ing
Seipin assembly; (3) changes in TAG lens nuclea ion: i TAG le els d op, Seipin
oligome iza ion can appea al e ed due o incomple e assembly. Thus, he s uc u al
indings in Li e al. may ep esen indi ec consequences a he han e idence o di ec
molecula in e ac ion.
7.7 Model G — Adig’s E ec s A ise om B own Adipose The mogenic
Regula ion
Adig dele ion educes he mogenic capaci y and al e s BAT unc ion. BAT LDs espond
apidly o he mogenic s a e, sh inking du ing sympa he ic ac i a ion and enla ging
when he mogenesis is supp essed. Thus, smalle LDs in Adig-KO mice could e lec
ele a ed BAT me abolic demand, while la ge LDs in Adig-o e exp essing adipose issue
could e lec supp essed he mogenesis. These sys emic e ec s could ully explain Li e
al.’s in i o indings wi hou in oking a di ec Seipin mechanism.
25
7.8 Summa y o Al e na i e Models
Toge he , hese models demons a e ha Li e al.’s in e p e a ion is only one o many
scien i ically plausible explana ions. A mo e conse a i e in e p e a ion would
acknowledge ha Adig likely in luences LD biology indi ec ly, Seipin binding is no
de ini i ely p o en, mul iple al e na i e pa hways could be e explain obse ed
pheno ypes, and issue speci ici y limi s gene al applicabili y.
8. Summa y o Key laws, Unce ain Claims and Recommended
Co ec ion Pa hs
Table 1 p o ides an in eg a ed, e idence-based e alua ion o he s uc u al,
biochemical, cellula , and physiological limi a ions iden i ied in he s udy “Adipogenin
p omo es he de elopmen o lipid d ople s by binding a dodecame ic seipin complex.”
The i s column summa izes key echnical weaknesses in da a acquisi ion, p ocessing,
and expe imen al design, including un alida ed Seipin s oichiome y, insu icien
s uc u al- esolu ion assessmen , lack o binding speci ici y con ols, and incomple e
me abolic pheno yping.
The second column ou lines claims ha a e no ully suppo ed by he p esen ed da a—
such as he physiological p edominance o he Seipin dodecame , he speci ici y and
s oichiome y o Adig binding, and he gene aliza ion o adipose- es ic ed mechanisms
o o he issues. The hi d column p oposes conc e e co ec i e s a egies and
alida ion pa hways, including symme y- ee c yo-EM econs uc ion, biophysical
quan i ica ion o Adig–Seipin a ini y, use o binding-de icien mu an escue assays,
lipidomic and me abolic lux measu emen s, and issue-speci ic alida ion ac oss non-
adipose LD- o ming cell ypes. Collec i ely, he able se es as a concise oadmap o
esol ing he mechanis ic unce ain ies and s eng hening he in e p e abili y and
physiological ele ance o he p oposed Adig–Seipin egula o y axis.
Table 1. Summa y o Key Flaws, Unce ain Claims, and Recommended Co ec ion
Pa hs
Ca ego y
Key Flaws / Limi a ions
Unce ain o
Unsuppo ed Claims
Recommended Co ec ion /
Valida ion Pa h
S uc u al
E idence
• C yo-EM map e ined wi h
en o ced C12 symme y; no
C1 econs uc ion shown.
• No biochemical alida ion
o Seipin s oichiome y
(na i e MS, SEC-MALS).
• “Dodecame ic
Seipin is he
physiological
p edominan o m.”
• “Adig binds wi h
de ined
• Gene a e symme y- ee
(C1) econs uc ions.
• Pe o m 3D a iabili y
analysis.
• Valida e oligome ic s a es
wi h na i e MS / SEC-MALS.
32
Re e ences
1 Choudha y, V. & Schnei e , R. Lipid d ople biogenesis om specialized ER
subdomains. Mic ob Cell 7, 218-221 (2020).
h ps://doi.o g/10.15698/mic2020.08.727
2 Olzmann, J. A. & Ca alho, P. Dynamics and unc ions o lipid d ople s. Na Re
Mol Cell Biol 20, 137-155 (2019). h ps://doi.o g/10.1038/s41580-018-0085-z
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