RESEARCH ARTICLE| CELL BIOLOGYOPEN ACCESS

Destabilization of β Cell FIT2 by saturated fatty acids alter lipid
droplet numbers and contribute to ER stress and diabetes
Xiaofeng Zhenga,b,c,1 , Qing Wei Calvin Hoa,1, Minni Chuaa, Olga Stelmashenkoa,b , Xin Yi Yeod,e , Sneha Muralidharanf, Federico Tortag
Elaine Guo Yan Chewa,h , Michelle Mulan Liana,h, Jia Nee Fooa,h , Sangyong Jungd,i, Sunny Hei Wonga, Nguan Soon Tana,j, Nanwei Tongc,
Guy A. Ruttera,k,l , Markus R. Wenkg, David L. Silverm , Per-Olof Berggrena,b,c,n,2, and Yusuf Alia,b,2,3,

Edited by Daniel Drucker, Mount Sinai Hospital, Toronto, ON, Canada; received July 16, 2021; accepted January 29, 2022

Western-type diets are linked to obesity and diabetes partly because of their high–saturated fatty acid (SFA) content. We found that SFAs, but not unsaturated fatty acids(USFAs), reduced lipid droplets (LDs) within pancreatic β cells. Mechanistically, SFAs,
but not USFAs, reduced LD formation by inducing S-acylation and proteasomal, mediated degradation of fat storage–inducing transmembrane protein 2 (FIT2), an endoplasmic reticulum (ER) resident protein important for LD formation. Targeted ablation of
FIT2 reduced β cell LD numbers, lowered β cell ATP levels, reduced Ca2+ signaling,
dampened vesicle exocytosis, down-regulated β cell transcription factors, up-regulated
unfolded protein response genes, and ﬁnally, exacerbated diet-induced diabetes in mice.
Subsequent mass spectrometry studies revealed increased C16:0 ceramide accumulationin islets of diet-induced diabetes mice lacking β cell FIT2. Inhibition of ceramide synthases ameliorated the enhanced ER stress and improved insulin secretion. FIT2 was
reduced in mouse diabetic islets, and separately, overexpression of FIT2 increased thenumber of intracellular LDs and rescued SFA-induced ER stress and apoptosis, thereby
highlighting the protective role of FIT2 and LDs against β cell lipotoxicity.

SigniﬁcanceWith obesity on the rise, there is a
growing appreciation for intracellularlipid droplet (LD) regulation. Here,
we show how saturated fatty acids(SFAs) reduce fat storage–inducing
transmembrane protein 2(FIT2)–facilitated, pancreatic β cell LD
biogenesis, which in turn induces βcell dysfunction and death, leading
to diabetes. This mechanism
involves direct acylation of FIT2
cysteine residues, which then marks
the FIT2 protein for endoplasmic
reticulum (ER)–associateddegradation. Loss of β cell FIT2 andLDs reduces insulin secretion,
increases intracellular ceramides,
stimulates ER stress, andexacerbates diet-induced diabetes inmice. While palmitate and stearate
degrade FIT2, unsaturated fatty acids
such as palmitoleate and oleate donot, results of which extend tonutrition and diabetes.

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FIT2 j lipid droplets j pancreatic β cells j ER stress j diet-induced diabetes

Numerous studies show that saturated fatty acids (SFAs), but not unsaturated fatty acids(USFAs), trigger endoplasmic reticulum (ER) stress, cell dysfunction, and apoptosis in a
myriad of cell types (1–4), including the pancreatic β cell. β cell vulnerability to free fatty
acids (i.e., lipotoxicity) reduces insulin provision and leads to diabetes (5). As with mosteukaryotic cells, β cells possess the ability to generate lipid droplets (LDs) from the ER (6).
Beyond mere storage containers for de novo–assembled neutral lipids and cholesterol esters(7–9), LDs were shown to act as hubs for innate immune defense, modulators of nuclear
function, and sites for protein storage and trafﬁcking (10–12). The number of LDs present
in a cell can dictate its energy rate, inﬂammation state, resistance to pathogens, ER stress,and cell death. LD biogenesis is a complex process involving many different ER resident
enzymes, including those that are involved in the synthesis of neutral lipids and cholesterolesters such as diacylglycerol transferases (13). However, LD budding from the ER is largely
regulated by a conserved tripartite ER protein machinery comprising of seipins, perilipins,
and fat storage–inducing transmembrane proteins (FITs) (14, 15). A modiﬁcation in eachof these proteins affect LD budding from the ER (13).
Despite considerable attention and the link to many diseases, the contribution of LDs to
β cell dysfunction and diabetes is just beginning to emerge. LDs are present in β cells, withnotable differences in numbers between rodent and human β cells and in increasing numbers
as β cells age (16). Prompted by our underpinning observation of a signiﬁcant change in LD
number between murine β cells exposed to SFAs with those exposed to USFAs, we lookedat how SFAs may directly inﬂuence LD numbers in β cells and, in turn, how this regulation
impacts β cell function and whole-body glucose homeostasis. We uncovered a mechanism bywhich SFAs reduce LD numbers, which involves the ubiquitously expressed FIT2/FITM2,
with subsequent impact on β cell function, survival, and overall glucose homeostasis. FIT2
belongs to a unique family of evolutionarily conserved ER resident, 6-transmembrane
domain, protein with its only other member being muscle cell–restricted FIT1/FITM1(17–21). FIT2, a critical protein for LD formation, has been shown to promote ER lipid
coalescence and LD formation, a function that is likely linked to its catalytic lipid phosphatase activity (15, 18, 22, 23). More recently, FIT2 was implicated in ER protein dysregulation, ER structure maintenance, and the maladaptive ER response in both yeast andmammalian cells (23, 24). Despite this, the role and importance of FIT2 in β cell LD formation, β cell function, and survival, especially during lipotoxicity, remains unknown.
Herein, we conﬁrmed the presence of FIT2 in β cells of mouse islets and show that palmitate induces proteasomal degradation of FIT2 through direct palmitoylation. This loss,
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Vol. 119No. 11e2113074119

Author contributions: X.Z., Q.W.C.H., S.J., and Y.A.
designed research; X.Z., Q.W.C.H., M.C., O.S., X.Y.Y., S.M.,
F.T., S.J., and Y.A. performed research; X.Z., O.S., S.M.,
F.T., J.N.F., N.T., D.L.S., P.-O.B., and Y.A. contributed new
reagents/analytic tools; X.Z., Q.W.C.H., M.C., O.S., X.X.Y.,
S.M., F.T., E.G.Y.C., M.M.L., J.N.F., S.J., S.H.W., N.S.T., N.T.,
G.A.R., M.R.W., D.L.S., P.-O.B., and Y.A. analyzed data; andG.A.R., M.R.W., D.L.S., P.-O.B., and Y.A. wrote the paper.
Competing interest statement: G.A.R. has received
grant funding from Les Laboratoires Servier and fromSun Pharmaceuticals and is a consultant for Sun
Pharmaceuticals. P.-O.B. is the founder of Biocrine AB
and is also the CEO of this company.
This article is a PNAS Direct Submission.
Copyright © 2022 the Author(s). Published by PNAS.
This open access article is distributed under Creative
Commons Attribution License 4.0 (CC BY).
1

X.Z. and Q.W.C.H. contributed equally to this work.

2

P.-O.B. and Y.A. contributed equally to this work.

3

To whom correspondence may be addressed. Email:yusuf.ali@ntu.edu.sg.
This article contains supporting information online at
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2113074119/-/DCSupplemental.
Published March 7, 2022.

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mimicked in mice with β cell–speciﬁc deletion of FIT2 (βFIT2KO),
resulted in reduced β cell LD numbers and diet-induced diabetes,with exacerbated glucose intolerance due to a reduced insulin secretory response. We also observed ceramide accumulation, a key driverof β cell ER stress, in islets from βFIT2KO mice, and inhibition ofceramide synthase improved insulin secretion in βFIT2KO islets.
Rescue of FIT2 in MIN6 cells, through overexpression, partiallyrestored LD biogenesis, reduced ER stress, and ameliorated SFAmediated β cell apoptosis (i.e., lipotoxicity). Beyond providing a conceptual advance in our understanding of how diet impacts the β cell,
this study highlights the restoration of LD formation as a potential
way of preserving normal β cell function in obesity.
Results

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Palmitate and Stearate Reduce β Cell FIT2 Protein Levels. To

recapitulate β cell vulnerability to SFAs, we ﬁrst exposed clonal
MIN6 cells (25) to physiologically relevant levels of either oleate (300 mM) or palmitate (300 mM) (26). Exposure to oleate
increased LD numbers, whereas exposure to palmitate did not(Fig. 1 A and B). Of the different LD regulatory proteins tested,
the expression of only FIT2 was signiﬁcantly reduced by palmitate(16:0) and stearate (18:0) compared with bovine serum albumin(BSA)–treated control cells. This reduction corresponded with asigniﬁcant increase in C/EBP homologous protein (CHOP/
DDIT3) protein levels, a major downstream proapoptotic arm of
the maladaptive ER stress response. FIT2 protein reduction was
not seen with chain-length, equivalent USFAs, palmitoleate(16:1), and oleate (18:1), in which levels, increased with USFA
treatment (Fig. 1C and SI Appendix, Fig. S1 A–D), in line withprevious observations with perilipin 2 (27). Seipin remained relatively unchanged after either SFA or USFA treatment. FIT2 protein was also reduced (threefold) in pancreatic islets from obeseand diabetic db/db mice, compared with their nondiabetic littermates (in which FIT2 messenger RNA [mRNA] colocalized withinsulin-positive, pancreatic β cells) (Fig. 1 D and E and SI
Appendix, Fig. S1E), supporting an earlier reported reduction in βcell LD numbers in diabetic mice (16).
Next, we generated MIN6 cells lacking FIT2 (Fit2 short
hairpin RNA [shRNA] cells) (SI Appendix, Fig. S1F) and micewith β cell FIT2 deletion (βFIT2KO) (SI Appendix, Fig. S1 G
and H). FIT2 down-regulation signiﬁcantly reduced LD number in both MIN6 cells (compared with scrambled, small interfering RNA [siRNA]–treated cells) (Fig. 1 F and G) and in
βFIT2KO mouse islets (compared with ﬂoxed control mouse
islets) (Fig. 1 H and I), the latter exacerbated by a moderate
SFA western diet (saturated fat accounts for 40% of totalenergy). Taken together, these results suggest that the loss of
FIT2 protein in β cells correlates with a failure to accumulateLDs, as observed with LDs in human β cells (16).

Loss of β Cell FIT2 Results in Mild Glucose Intolerance. Given

that β cell FIT2 levels were lowered following SFA exposure,
we determined whether this change might impact whole-body
glucose homeostasis. Here, we focused on 12-wk-old malemice. Concerns of RIP-Cre “leakiness” (28) was addressed byshowing no signiﬁcant change in Fit2 mRNA expression in
βFIT2KO extrapancreatic tissues (SI Appendix, Fig. S1I). There
were no changes in intraperitoneal glucose tolerance test
(IPGTT) and insulin tolerance test (ITT) in the control mouselines, except for the well-documented glucose excursion increase
in RIP-Cre mice (29), and yet, comparatively, 12-wk-old
βFIT2KO mice displayed a mild, but signiﬁcant, glucose intolerance compared with the line with the Cre transgene alone(Fig. 2 A and B).

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The poorer glycemic control in βFIT2KO mice was associatedwith a signiﬁcantly muted insulin response to high glucose, bothin vivo and ex vivo (Fig. 2 E and F) and not by changes in peripheral insulin sensitivity (as measured by insulin tolerance test) (Fig.
2 C and D). There were no discernible differences in islet morphology, composition, pancreas/islet insulin content, and insulin
granule morphology between βFIT2KO and ﬂoxed control mice
(SI Appendix, Fig. S2 A–F). Rather, the muted insulin response in
βFIT2KO islets could be partly attributed to an observed reduction in glucose-induced ATP/ADP ratio and a blunted, glucosestimulated intracellular Ca2+ inﬂux (SI Appendix, Fig. S2 G–I).
Interestingly, diminished insulin secretion (Fig. 2 F and H) andCa2+ inﬂux response (SI Appendix, Fig. S2 H and J), following
KCl-induced membrane depolarization as well as reduced exocytosis (measured by membrane capacitance), were also observed in
βFIT2KO islets (SI Appendix, Fig. S2 K and L). This suggeststhat mechanisms independent of glucose metabolism may additionally contribute toward the observed reduction in insulin secretion from β cells devoid of FIT2. Comparative RNA-sequencing(RNASeq) and gene ontology (GO) analysis between islet cells
from 12-wk-old chow diet–fed βFIT2KO and ﬂoxed control
mice revealed distinct changes in gene clusters (Datasets S1 andS2) related to 1) vesicle fusion and exocytosis, 2) ATP binding, 3)
Ca2+ transport, and 4) ER-related genes, as well as 5) a signiﬁcantdown-regulation of transcription factors linked to β cell function(Fig. 2I and Datasets S3 and S4). Changes in key β cell function/
identity genes in islets from βFIT2KO, such as Pdx1 and MafA,
were corroborated using real-time qPCR (SI Appendix, Fig. S2M).
Overall, these results support the view that impaired glucose tolerance in βFIT2KO mice is driven by reduced β cell function.
Loss of FIT2 Exacerbates Diet-Induced Diabetes and Increases
β Cell Ceramides and ER Stress. Next, we metabolically chal-lenged, 5-wk-old βFIT2KO mice with a high–saturated fat,high-sucrose–containing diet (western diet) for a further 25 wk.
Plasma glucose concentrations following intraperitoneal bolus glucose (2 g/kg) increased in 30-wk-old, chow-fed mice, and this was
signiﬁcantly exacerbated in aged-matched western diet–fed
βFIT2KO mice, suggesting glucose intolerance (Fig. 3 A and B).
Although western diet–fed mice displayed insulin resistance, there
were no signiﬁcant differences between βFIT2KO mice and ﬂoxed
control mice within each diet group (Fig. 3 C and D). While nosigniﬁcant changes in fasting-state blood glucose levels wererecorded, the fed-state blood glucose levels were signiﬁcantly
higher in western diet–fed βFIT2KO (βFIT2 knockout [KOWest]) mice compared with the ﬂoxed control mice (FIT2
ﬂoxed [FL West]) (Fig. 3E). Hyperinsulinemia is a wellestablished phenotype of diet-induced diabetes, and both western diet–fed, ﬂoxed control (FL West) and βFIT2KO (KOWest) mice displayed hyperinsulinemia (Fig. 3F). Fasting
plasma insulin levels were, however, observed to be modestly
but signiﬁcantly lower in βFIT2KO (KO West) mice, a difference that was more pronounced (and signiﬁcant) following a
glucose challenge. Hence, the loss of FIT2 in β cells did significantly compromise compensatory hyperinsulinemia, and while
this was not reﬂected in fasting glucose levels, it was in linewith the signiﬁcantly elevated, fed state glucose levels of western diet–fed βFIT2KO (KO West) mice. Noteworthy, weobserved no change in plasma insulin levels 15-min postglucose administration in all western diet–fed mice. Such muted,
glucose-stimulated insulin responses in vivo have been
described in hyperinsulinemic mice (29). The exacerbated βcell dysfunction in βFIT2KO mice exposed to a western
diet was further scrutinized. We observed higher levels ofpnas.orgAC

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Fig. 1. FA-mediated changes in β cell LD numbers relates to FIT2 protein abundance. (A) BODIPY staining of LDs in MIN6 cells treated with BSA, oleate, or
palmitate (300 mM) for 24 h. LDs are stained with BODIPY (green) and nuclei stained with DAPI (blue). Images shown are maximum intensity projections.
(Scale bar, 50 mm.) (B) Quantitation of BODIPY-positive puncta per cell using Image J. (>500 cells from three independent experiments were analyzed pergroup). (C) Representative immunoblot of several LD formation proteins after MIN6 cells were treated with 300 mM palmitoleate (16:1), oleate (18:1), palmitate (16:0), stearate (18:0), or BSA (control) for 24 h. (D and E) Representative immunoblot and semiquantitation of FIT2 protein in islets isolated from db/+
and db/db mice (16-wk-old male, n = 4). (F) BODIPY staining of LDs in control (Ctrl shRNA) or FIT2 knockdown (FIT2 shRNA) MIN6 cells treated with BSA or
300 mM oleate for 48 h. LDs were stained with BODIPY (green), and nuclei were stained with DAPI (blue). Images shown are maximum intensity projections.
(Scale Bar, 50 mm.) (G) Quantitation of BODIPY-positive punctae per cell using Image J. BSA-treated control (Ctrl shRNA, blue) or FIT2 knockdown (FIT2 shRNA,red) and oleate-treated (300 mM) control (Ctrl shRNA, green) or FIT2 knockdown (FIT2 shRNA, purple) (>1,000 cells from three independent experiments
were analyzed per group). (H) BODIPY staining of LDs in pancreas sections of chow-fed, ﬂoxed control (FL, blue) and βFIT2KO (KO, red) and western diet(West, 25 wk)–fed, ﬂoxed control (FL, green) and βFIT2KO (KO, purple). LDs were visualized using a BODIPY stain (green). β cells were stained with insulin(red) and nuclei stained with DAPI (blue). Images shown are maximum intensity projections. (Scale bar, 25 mm.) (I) Quantitation of BODIPY-positive puncta
per cell using Image J (n = 3, ≥9 islets from three mice were analyzed per group). Values shown are mean 6 SEM; *P < 0.05; **P < 0.01; and ***P < 0.001
 ak post hoc test).
(B: one-way ANOVA with Tukey’s post hoc test, D: two-tailed Student's t test, and G and I: two-way ANOVA with Sid

p-IRE1a+/insulin+ cells and ATF4+/insulin+ cells in western
diet–fed βFIT2KO (KO West) mice compared with the ﬂoxed
control mice (FL West) (Fig. 3 G–J), suggesting increased,maladaptive ER stress responses in vivo. As ER stress is an
important driver of β cell dysfunction (30, 31), we determined
whether β cell FIT2 and LDs played a role in the palmitateinduced ER stress response. p-IRE1a was signiﬁcantly elevated, with a time-dependent increase in IRE-1, ATF4, and
CHOP protein levels in palmitate-treated FIT2 shRNA cells,
compared with control cells (Fig. 3K and SI Appendix, Fig. S3
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A–D). The ampliﬁed ER stress response (ATF4, CHOP, andIRE-1 pathways) coincided with reduced insulin secretion (SIAppendix, Fig. S3E), increased cell death (Fig. 3 L and M),
and a signiﬁcantly elevated activity of caspase-3/7, suggesting
increased apoptosis in FIT2 shRNA cells (Fig. 3N). Separately,
liquid chromatography–mass spectrometry (LC-MS) analysis
of ceramide levels in pancreatic islets isolated from KO Westand FL West (fed) mice revealed accumulation of toxic C16
ceramide (Cer d18:1/16:0) in the βFIT2KO islets (Fig. 3O).
Similarly, there was a modest increase in the ceramide
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KO
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Fig. 2. Glucose homeostasis and β cell function is impaired in βFIT2KO mice. Intraperitoneal glucose tolerance test (GTT) of age-matched, chow-fed, ﬂoxed control(FL), βFIT2KO (KO), rat insulin II promoter Cre recombinase (RIP-Cre), and wild-type (WT) mice (12- to 16-wk-old male, n = 10 to 13) (A) and corresponding area under
the curve (AUC) of blood glucose levels from A (B). Insulin tolerance test (ITT) on FL, KO, RIP-Cre, and WT mice (12- to 16-wk-old male, n = 7) (C) and correspondingarea under the curve (AUC) of blood glucose levels from C (D). (E) In vivo insulin secretion following intraperitoneal glucose administration in ﬂoxed control (FL) and
βFIT2KO (KO) mice (12-wk-old male, n = 7). (F) Ex vivo islet glucose-stimulated insulin secretion from ﬂoxed control (FL) and βFIT2KO (KO) mice (12-wk-old male,n = 3). AUC for insulin secretion at 16.7 mM glucose (n = 3) (G) and 25 mM KCl from F (H). (I) GO-clustering analysis of down- and up-regulated genes in (βFIT2KO)
KO as compared with ﬂoxed control (FL) islets (n = 5 mice). Enrichment score (≥1.3) of each GO cluster is indicated in parenthesis below each respective term, andﬁve highly signiﬁcant altered genes for each GO cluster are listed below. The full gene list is provided in Datasets S1 and S2. Values shown are mean 6 SEM;
*P < 0.05; **P < 0.01; and ***P < 0.001 (B and D: one-way ANOVA with Tukey’s post hoc test and E, F, G, and H: two-tailed Student's t test.

precursor C16 dihydroceramide (Cer d18:0/16:0) (P = 0.055)
and in other dihydroceramides containing saturated fatty acyl
chains, suggesting enhanced de novo ceramide synthesis in islets
lacking FIT2. Accumulation of ceramides in β cells was corroborated
by immunoﬂuorescence (IF) analysis in βFIT2KO mice (SIAppendix, Fig. S3 F and G), and cotreatment of palmitate withfumonisin B1 (FB1), a pharmacological inhibitor of ceramide synthases, improved insulin secretion (Fig. 3P) in islets from βFIT2KO
mice and reduced CHOP levels in palmitate-treated MIN6 cells(Fig. 3 Q and R) (32). Together, these data suggest that ceramide
accumulation contributes to enhanced ER stress and dysfunction inwestern diet–fed βFIT2KO mice.

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Partial Restoration of FIT2 Levels Rescues LD Biogenesis and
Mitigates Palmitate-Mediated Effects in MIN6 Cells. The roleof FIT2 loss in SFA-induced β cell apoptosis was further investigated through its overexpression in MIN6 cells. Cells were transiently transfected with either an expression vector–encoding FIT2
(pcDNA3.1-FIT2) or with a control vector (pcDNA3.1-mock)prior to palmitate (or BSA) exposure. FIT2 levels increased byapproximately threefold in FIT2-overexpressing (FIT2-OE) cells
treated with BSA compared with control (mock cells) (Fig. 4 A
and B). While palmitate exposure resulted in an ∼95% reductionof FIT2 in control cells (mock transfected), this was partially attenuated in FIT-OE cells (∼50% reduction) (Fig. 4 A and B). This
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Pal

37
BSA
-

Pal
-

Relative Intensity(CHOP/β-actin)

Stimulation Index

Ctrl FIT2***
pmol/IEQ***

FIT2 shRNA**

O
6
Casp-3/7 activity (fold)

Pal

N
15

0
shRNA

Stimulation Index

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BSA

PI-positive cells (% of total)

PI/DAPI

FB1

KO westkDa

Ctrl shRNA

P 2.02.0

KO

West

IRE1α

00

st

K

Ctrl shRNA

FL

***

1010

Chow West

L

KO

Chow

FIT2

FL

st

FL

K

J

FL

w

e
e
w FLw KOFLO choKO
O
FL
K

0

120

FL chow15
15

Time(min)
(min)
Time

K
O

ow

AUC of ITT (fold)

Blood glucose (mM)

es
t

11

es
t

es
t

FL

es
t

FLWestWest
FL
KOWest
West
KO

22

K
O

22

ch

KOChow
Chow
KO

**

33

KO

1

G

ATF4/INS/DAPI

44

FL

60
90
Time (min)

2

1515

66

00

30

FLChow
Chow
FL

West

p-IRE1 fluorescence intensity (fold)

88

0

KO

I

***

0

*

4

W

W

ho
w

C

FL

2

West

ATF4 fluorescenceintensity (fold)H
p-IRE1α fluorescenceintensity (fold)

Chow

K
O

FL

C

ho

w

KO

FL

4

p-IRE1α /INS/DAPI

PlPlasma
asma iinsulin
nsulin(µg/ml)(ng/ml)Fed-stateFed
State
GlucoseBBloodlood G
lucose(mM)
(mM)

***

KO

Chow

6

W

FL

15
15

FL

00

120
120

KO

60
90
60
90
Time (min)
Time
(min)

Ch
ow

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30

3
FL Chow
KO Chow
FL West
KO West

ATF4 fluorescence intensity (fold)

E

00

FL

00

11

W

1010

22

D

8

KO

2020

***

Ch
ow

3030

C 10

***

33

AUC of IPGTT(Fold Change)

FL Chow
FL Chow
KO Chow
KO Chow
FLFLWest
West
KOKOWestWest

AUC of GTT (fold)

Blood glucose (mmol/L)

Blood glucose (mM)

B
4040

FL

A

***

2

1

Pal
+
0

BSA Pal
-

Pal
+

FB1

BSA
-

Pal
-

Pal
+

Fig. 3. Loss of β cell FIT2 exacerbates ER stress and survival. Intraperitoneal glucose tolerance test on chow-fed, ﬂoxed control (FL, blue) and βFIT2KO (KO, red)or western diet (West)–fed, ﬂoxed control (FL, green) and βFIT2KO (KO, purple) (30-wk-old male, n = 5) (A) and area under the curve (AUC) of blood glucose levels
from A (B). ITT on chow-fed, ﬂoxed control (FL, blue) and βFIT2KO (KO, red) or western diet (West) –fed, ﬂoxed control (FL, green) and βFIT2KO (KO, purple) (30-wkold male, n = 4) (C) and AUC of blood glucose levels from C (D). (E) Fed state blood glucose levels of chow-fed, ﬂoxed control (FL, blue) and βFIT2KO (KO, red) or
western diet (West) –fed, ﬂoxed control (FL, green) and βFIT2KO (KO, purple) (30-wk-old male, n = 5). (F) In vivo insulin secretion following glucose intraperitoneal
administration in chow-fed, ﬂoxed control (FL Chow, blue) and βFIT2KO (KO Chow, red) or western diet (West) –fed, ﬂoxed control (FL West, green) and βFIT2KO(KO West, purple) (30-wk-old male, n = 5). (G and I) Representative immunostain for p-IRE1a and ATF4 (green), insulin (red), and DAPI (blue) in pancreas sections
from ﬂoxed control (FL) and βFIT2KO (KO) mice treated with either chow or western diet (30-wk-old male). Images shown are maximum intensity projections.
(Scale bar, 50 mm.) (H and J) Quantitation of relative ﬂuorescence intensity of p-IRE1a and ATF4 staining within insulin-positive cells (n = 3 to 5 mice, ≥9 islets fromeach mouse were analyzed per group). (K) Representative immunoblot of indicated proteins in control shRNA-transfected or FIT2-stable knockdown MIN6 cells(FIT2 shRNA) in the absence or presence of palmitate (300 mM). (L) Representative image of control (Ctrl) shRNA-transfected or FIT2-stable knockdown (FIT2 shRNA)
MIN6 cells in the absence (BSA only) or presence of palmitate (300 mM, 24 h) stained for DAPI (blue) and propidium iodide (PI, red). Images shown are maximum
intensity projections. (Scale bar, 50 mm.) (M) Quantitation of the percentage of PI-positive cells over total number of cells (>1,000 cells from three independent
experiments were analyzed per group). (N) Measurements of capase-3/7 activity in control shRNA-transfected or FIT2 knockdown (FIT2 shRNA) MIN6 cells in theabsence (BSA only) or presence of palmitate (300 mM, 24 h) (n = 4). (O) Quantitation of different ceramide species in islets from western diet–fed, ﬂoxed mice (FLWest) and βFIT2KO (KO West) (30-wk-old male) by LC-MS expressed as picomole per islet equivalent (pmol/IEQ) (n = 3). (P) Ex vivo insulin secretion, represented
as stimulation index (16.7 mM/2.8 mM), from βFIT2KO islets treated with FB1 and palmitate. KO islets were pretreated with FB1 (10 mM) for 2 h, followed bypalmitate (300 mM) in presence of FB1 for 24 h (ﬁve islets from three different mice; n = 3 independent experiments). (Q and R) Representative immunoblot
and analysis of CHOP in MIN6 cells treated with FB1 (10 mM) in the presence and absence of palmitate (300 mM, 24 h) (n = 4). Values shown are mean 6 SEM;
*P < 0.05; **P < 0.01; and ***P < 0.001 (P and R: one-way ANOVA with Tukey’s post hoc test, F and O: two-tailed Student's t test [between 15 and 0 min, FL West a
k post hoc test.
and KO West, respectively]), and B, D, E, H, J, M, and N: two-way ANOVA with Sid

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suggests that overexpression of FIT2 partially compensated forpalmitate-induced reduction in FIT2 protein levels. This partial
restoration of FIT2 in FIT-OE cells exposed to palmitate led to asigniﬁcant increase in the number of LDs (Fig. 4 C and D), suggesting a partial LD rescue and that β cells LDs can be formed following palmitate exposure provided FIT2 is preserved. This rescue
corresponded with a reduction in CHOP/DDIT3 protein levels
and lowered caspase-3/7 activity in FIT2-OE cells exposed to palmitate (Fig. 4 E–G). Together, these results suggest that partial
compensation of FIT2 loss ameliorates palmitate-induced lipotoxicity in β cells.

Downloaded from https://www.pnas.org by 116.14.152.5 on March 7, 2022 from IP address 116.14.152.5.

Palmitoylation and ERAD-C Contribute to FIT2 Protein Loss.

We next sought to elucidate the mechanism(s) that may explain
SFA-induced FIT2 loss. The observed, SFA-mediated down-regulation of FIT2 in MIN6 cells was not seen at the transcriptional level(SI Appendix, Fig. S4A) but was abrogated in the presence of proteasome inhibitor MG132 (Fig. 5 A and B), suggesting that SFAs
modulate FIT2 posttranslational stability. Given that FIT2 is an
ER resident protein, we assessed involvement of the ER-associateddegradation (ERAD) pathway. The ERAD inhibitor eeyarestatin I(33) partially, but signiﬁcantly, attenuated palmitate-induced FIT2
loss (Fig. 5 C and D). Increased association between FIT2 andmembrane-associated ring-CH–type ﬁnger 6 (MARCH6), a mammalian E3 ligase complex responsible for degradation of the
ERAD-cytosolic substrate (ERAD-C) (34), was observed in MIN6
cells treated with palmitate (Fig. 5E). Furthermore, MARCH6
down-regulation (∼50%; SI Appendix, Fig. S4B), but not autocrine
motility factor receptor (AMFR) down-regulation, an E3 ubiquitin
ligase linked to ERAD-luminal substrate (ERAD-L) pathway (SIAppendix, Fig. S4 C–E), signiﬁcantly rescued palmitate-mediated
FIT2 loss (Fig. 5 F and G). Taken together, these results suggest
ERAD-C, but not ERAD-L, contributes to palmitate-mediated
FIT2 degradation.
We next tested the possibility that palmitate may directly
modify FIT2, given that an in silico analysis (GPS–lipid) (35)showed high probability of fatty acid S-acylation taking place atonly four cysteine sites (Cys-7, Cys-251, Cys-140, and Cys-70)(SI Appendix, Fig. S4F). S-palmitoylation on FIT2 protein was
conﬁrmed using two separate, distinct chemistry S-palmitoylation
assays, with higher levels of S-acylated FIT2 proteins detected
in MIN6 cells exposed to palmitate (Fig. 5H and SI Appendix,
Fig. S4 G and H. Mutating all four predicted S-acylation sites(Cys to Ala) abrogated palmitate-induced modiﬁcation understeady-state (BSA) and exogenous palmitate conditions (Fig. 5H).
To conﬁrm palmitoylation led to FIT2 degradation, cerulenin (apalmitoylation inhibitor) (36) was added together with palmitate.
Cerulenin signiﬁcantly reversed palmitate-mediated FIT2 protein
loss (Fig. 5 I and J).
Given potential off-target effects of cerulenin (37), we tested
the stability of FIT2 mutants (C7/251/140/70A) in the presence and absence of palmitate. Single mutation of either cysteine residue tended to abrogate palmitate-induced FIT2 protein
loss (SI Appendix, Fig. S4 I and J). However, mutation of all
four cysteine residues completely blocked palmitate- (Fig. 5 K
and L) as well as stearate- (SI Appendix, Fig. S4 K and L) mediated FIT2 degradation.
Discussion
Here, we provide evidence of a fatty acid (FA)–type, speciﬁc
modulation of an ER resident, LD formation-linked protein, or
FIT2 in β cells. Our results show that LD numbers are affectedby the types of FA to which cells are exposed and that one

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https://doi.org/10.1073/pnas.2113074119

pathway responsible for differential toxicity between dietary SFA
and USFA (in the form of palmitate and oleate, respectively)
involves cellular FIT2 protein stability and the consequent
ability of β cells to accumulate LDs. We present a potential
mechanism through which SFAs reduce FIT2 levels. Direct palmitoylation of FIT2 lead to increased β cell ER stress response,
reduced insulin secretion, and elevated β cell apoptosis, as wellas increased susceptibility to diet-induced diabetes. As FIT2
overexpression was able to rescue some LD formation and attenuate palmitate-induced ER stress, we highlight the ﬁnding thatthe β cell’s ability to withstand lipotoxicity is contingent on its
capacity to maintain FIT2 and to generate LDs. Future studies
will, however, be needed to explore the effects of FIT2 overexpression in vivo.
LDs are an important source for organelle membrane lipids
and proteins (38). Mitochondrial lipid uptake from ER is essential
for maintaining mitochondrial membrane integrity, which may beaffected by the loss of FIT2 consistent with decreased [ATP]/[ADP] ratios (in βFIT2KO islet cells) (39). In addition to reduced
cellular ATP, lowered intracellular Ca2+ inﬂux was also noted inboth high-glucose and high-K+–induced conditions, suggesting
metabolic, independent changes because compromised Ca2+
channel activity and vesicle exocytosis (conﬁrmed by cell capacitance recordings) were recorded in the absence of high glucose.
Indeed, RNASeq analysis conﬁrmed that, apart from reduced
genes linked to carbohydrate metabolism, there were also reduction of genes linked to β cell transcription factors, vesicle fusion,and exocytosis as well as Ca2+ channel transport. The reduced
expression of β cell transcription factors, such as Pdx1 (pancreaticand duodenal homeobox 1) and MafA (V-Maf avian musculoaponeurotic ﬁbrosarcoma oncogene homolog A), allude to a loss of βcell identity, a characteristic of ER-stressed T2D islets (40). The
expression of Kcnb1 (voltage-dependent K+ channel Kv2.1), Syt7
(synaptotagmin VII), and Rab1b (member of RAS oncogene family) were also lower in islets of βFIT2KO mice compared with FL
mice. Both KCNB1 and SYT7 play key roles in insulin exocytosis,
and their depletion led to impaired insulin vesicle trafﬁcking andsecretion in β cells (41, 42). RAP1b is a small GTP-binding protein linked to accentuated insulin exocytosis by GLP-1 and glibenclamide coadministration (43). Syn2 (syntaxin-2) expression was
also lower in βFIT2KO islet cells (versus FL islet cells), but incontrast to KCNB1, SYT7, RAP1b, and all other known β cellsyntaxins (e.g., SYN1A, SYN3, and SYN4), a reported loss of
SYN2 increased exocytotic events in β cells (44). The action of
SYN2 suggests redundancy of syntaxins in SNARE complexes.
The impact of FIT2 and LD loss on β cell transcription factors
and vesicle exocytosis require further investigation, especially since
LDs were also shown to regulate nuclear translocation and endosomal trafﬁcking (45).
We also observed signiﬁcant elevation of ER stress–related proteins, such as p-IRE1a and ATF4 in FIT2KD cells, and in isletsof βFIT2KO mice. Corroborating transcriptomic evidence of ER
protein and REDOX gene up-regulation in βFIT2KO islets suggests that loss of LDs induces the maladaptive ER stress responsein β cells. Accumulation of C16:0 ceramides in islets of
βFIT2KO mice may also contribute to the observed ER stressand β cell dysfunction, though whether these result from changes
in circulatory ceramide levels remains to be established. Nevertheless, a GENEVESTIGATOR search revealed CerS5 and CerS6 tobe the most abundant ceramide synthases in pancreatic islets (46).
CerS5 and CerS6 generate C16 and C14 chain-length ceramides
and are likely responsible for the observed difference in C16:0 ceramide levels in western diet–fed βFIT2KO islets and ﬂoxed islets(47, 48). Further inhibition of ceramide synthesis by FB1
pnas.org

A

kDa

FIT2

BODIPY/DAPI

C

BSA

Pal

FIT2-OE***

33(FIT2/ -actin)

22
*

11
00

**

8
6
4
2
0

Mock FIT2-OE Mock FIT2-OE
Mock FIT2-OE Mock FIT2-OE
BSA
Pal

BSA

kDa

CHOP

25
β−actin37
Mock FIT2-OE Mock FIT2-OE
BSA

Relative Intensity(CHOP/β -actin)

F
E

***

3
2
1
0

Pal

BSA

Pal

Mock FIT2-OE Mock FIT2-OE
BSA

Pal

G

Casp-3/7 activity (fold)

Relative Intensity
Relative Intensity(FIT2/β-actin)

B

Mock

37
Mock FIT2-OE Mock FIT2-OE

BODIPY-positive puncta/Cell20

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D

Pal

***

2.5
2.0
1.5
1.0
0.5
0.0

Mock FIT2-OE Mock FIT2-OE
BSA

Pal

Fig. 4. Overexpression of FIT2 in MIN6 cells ameliorates palmitate-induced lipotoxicity. (A–G) MIN6 cells were cultured in 6-well plates and transfected with
0.5 mg empty pcDNA3.1 vector (Mock) or pcDNA3.1-FIT2 (FIT2-OE) per well, followed by treatment with BSA or palmitate (300 mM) for 24 h. (A and B) Representative immunoblot and semiquantitation FIT2 protein levels (n = 3 independent experiments). (C) LD staining (BODIDY, green) in vector-transfected(Mock) or in FIT2-overexpressing (FIT2-OE) MIN6 cells (nuclei stained with DAPI; blue) in the absence (BSA) and presence of palmitate (300 mM, 24 h). Images
shown are maximum intensity projections with a scale bar of 50 mm. (D) Quantitation of BODIPY-positive puncta per cell using Image J. (>1,000 cells fromthree independent experiments were analyzed per group). Representative immunoblot analysis (E) and corresponding semiquantitation of CHOP levels (n =4) (F). (G) Measurements of capase-3/7 activity in control vector-transfected (Mock) or FIT2-overexpressing (FIT2-OE) MIN6 cells in the absence (BSA) or pres ak post hoc test).
ence of palmitate (300 mM) (n = 4). Values shown are mean 6 SEM; *P < 0.05; **P < 0.01; and ***P < 0.001 (two-way ANOVA with Sid

ameliorated palmitate-mediated ER stress in MIN6 cells andimproved insulin secretion in βFIT2KO islets. When coupledwith increased C16:0 ceramides levels in islets that lack FIT2, thedata suggests that increased ceramides link β cell FIT2 loss withenhanced ER stress and reduced β cell function, adding to current
knowledge of FIT2 loss, leading to ER lipid imbalance and ER
dysregulation (23, 24).
We describe here a pathway through which palmitate drives
ER stress in β cells, with FIT2 stability and LD accumulation
taking center stage. Failure to sequester lipids away from the
ER is likely to increase the levels of substrates for the
ER-localized enzymes serine palmitoyltransferase and ceramide
synthase, thus increasing ER ceramide levels (49, 50). Ceramide
accumulation has been suggested to alter ER membrane properties, reduce ER protein export, disrupt ER Ca2+ homeostasis,
and increase ER stress–mediated apoptosis (51, 52). The latter
was conﬁrmed by increased presence of CHOP, a known preapoptotic death signal, in MIN6 cells and in islets lacking FIT2.
Thus, under lipotoxic conditions, FIT2 and LDs perhaps function critically to sequester and store FAs in their less toxic,
esteriﬁed forms thereby preventing ceramides, especially C16:0,
from accumulating within the ER.
When LDs are allowed to accumulate in β cells, for example
through desnutrin (a triacylglycerol hydrolase) ablation, a similar, blunted glucose-stimulated insulin secretion (GSIS)
response was observed (53). These results may seem contradictory to the current ﬁndings. However, a signiﬁcant driver for
dysfunction in the desnutrin knockout mice is reduced fatty
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Vol. 119No. 11e2113074119

acid utilization by mitochondria, rather than enhanced β cell ER
stress seen in our FIT2 ablation model. So while LD formationand lipid sequestration away from the ER mitigates lipotoxicity,
this is perhaps based on the assumption that LD utilization isunaffected. Here, further work is required to delineate thedynamics of LD accumulation, utilization, and turnover, especially since a higher number of LDs has been observed in humandiabetic β cells (16). Nevertheless, it is increasingly clear that theformation of LDs, especially during lipotoxic conditions, is critical for preventing ER lipid accumulation and ER stress.
A fundamental mechanism governing LD loss is the posttranslational modiﬁcation of FIT2 by palmitate (and stearate) and its
subsequent degradation in β cells. This may explain the absenceof FIT2 in genome-wide association or islet RNASeq-related
studies. However, a recent type 2 diabetes meta-analysis study
identiﬁed an East Asian diabetes-associated loci (RS6017317)
directly in the regulatory region of FIT2 (54), and FIT2 lies
adjacent to the pernicious and well-described HNF4a loci. The
molecular switch of this protein, and a potential therapeuticstrategy, likely involves maintaining its protein stability at the
ER. Palmitoylation commonly occurs on transmembrane proteins, affecting protein stability and localization (55, 56). Interfering with palmitoylation either pharmacologically or throughsite-directed mutagenesis reduced palmitate-induced FIT2 degradation, and a similar mechanism was reported in other transmembrane proteins, such as TBC1D3 and CDCP1 (57, 58).
We further identiﬁed ERAD-C as a contributing pathway to
FIT2 degradation. We found increased MARCH6 and FIT2
https://doi.org/10.1073/pnas.2113074119

7 of 10β−actin

37

1.0
1.0

BSA Pal Pal
+

MG132

E

0.0
0.0

BSA
MG132
-

IP: 6xHis

Pal
-

0.5
0.5

ES

Pal
+

37
BSA Pal Pal
+

kDa
37

G
kDa

FIT2

IB: FIT220β−actin25

IB: β−actin

37
BSA Pal BSA Pal
6xHis

37
Ctrl March6
siRNA siRNA

Ctrl March6
siRNA siRNA

BSA

Pal

BSA PalIsotype IgG
Control

H

FIT2
1: IF
kDa 2: cUF
3: cBF
25 4: pUF
5: pBF

WT
C7/70/
140/251A
3

4

5

1

3

2

BSA

L
kDa
β−actin
BSA BSA Pal Pal
WT++
C7/70/140/251A +
+

5

-

0.6

Pal
Pal Pal+ES
Pal

-

+

*

0.4
0.2

0.0

J
kDa
FIT2

20

β−actin

37

2.0

2.0
1.5

1.5

*

1.0

1.0

0.5

0.5

0.0

0.0

BSA

Cerulenin

BSA-

PalPal+Cer

Pal
-

Pal
+

Pal

K
His-FIT2

4

25
37

Fold Change in FIT2
protein (Pal/BSA)

2

BSA
BSA

Ctrl March6*
siRNA siRNAI

BSA Pal Pal
Cerulenin +

25
1

0.0
0.0

ES

F

Input

IB: MARCH6

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20

β−actin

0.5
0.5

Relative Intensity(FIT2/β-actin)

20

**

1.0
1.0

kDa

FIT2

1.5
1.5

Fold Change in FIT2
protein (Pal/BSA)

Relative Intensity(FIT2/β-actin)

FIT2

***

1.5
1.5

kDa

D

C

2.0
2.0

Relative Intensity(FIT2/β-actin)

B

A

**

1.5
1.0
0.5

0.0
WT
C7/70/140/251A+-+

Fig. 5. Palmitate-induced reduction of FIT2 protein involves palmitoylation with ERAD-C contributing to its degradation in MIN6 cells. (A–D) Representative
immunoblot analysis and semiquantitation of FIT2 protein in MIN6 cells pretreated with respective pharmacological inhibitors 10 mM MG132 or 10 mM Eeyarestatin (ES) for 2 h, followed by cotreatment with inhibitors in the presence of palmitate (300 mM) for 4 h (n = 3). (E) Representative immunoblot (IB) for
coimmunoprecipitation (IP) of FIT2 and MARCH6 in MIN6 cells in the absence (BSA) and presence of palmitate (Pal, 300 mM, 6 h), following pretreatment
with 10 mM MG132 for 2 h. Pull-down of FIT2 using equal amount of input protein was carried out using 6×His antibody (with isotype control), and thereafter, captured proteins were probed for MARCH6 protein using the tGFP antibody. Representative immunoblot analysis (F) and semiquantitation of FIT2 protein from serum-starved MIN6 cells (2 h) transfected with scrambled control siRNA (Ctrl siRNA) or siRNA against March6 (March 6 siRNA), followed by treatment with BSA or palmitate (300 mM) for 4 h (n = 4) (G). (H) Representative immunoblot of S-palmitoylation assay on both wild-type (WT) and mutant (C7/70/
140/251A) FIT2 under steady-state (BSA) and palmitate (Pal, 300 mM, 6 h) conditions. The increased S-palmitoylation of FIT2 under palmitate conditions (lane3, CAPTUREome resin cleaved bound fraction [cBF]) was markedly abrogated in the mutant FIT2. Input fraction (IF) and the ﬂowthrough fraction which represents the cleaved unbound fraction (cUF) were collected and analyzed. A separate set of protein lysates (negative control) was treated with acyl-preservation
reagent and subjected to incubation with CAPTUREome resin (ﬂowthrough fraction was used as the preserved unbound fraction [pUF], while eluted fraction
was used as the preserved bound fraction [pBF]). Representative immunoblot (I) and corresponding semiquantitation of FIT2 protein levels in MIN6 cells pretreated with cerulenin (45 mM) for 2 h, followed by cotreatment of cerulenin (45 mM) in the presence of palmitate (300 mM) for 4 h (n = 3) (J). Representative
immunoblot analysis (K) and corresponding semiquantitation of FIT2 protein levels in MIN6 cells transfected with pcDNA3.1-FIT2/V5-His (WT) or mutant (C7/70/140/251A), followed by treatment with BSA or palmitate (300 mM) for 24 h (L). Values shown are mean 6 SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. B,
D, and J: one-way ANOVA with Tukey’s post hoc test and G and L: two-tailed Student's t test.

protein association in the presence of palmitate and reduced
FIT2 degradation in MARCH6 knockdown cells, but not in
AMFR (ERAD-L) knockdown cells. Mutating predicted,
S-acylated cysteine residues abrogated FIT2 palmitoylation anddegradation. These results suggest that SFA-mediated S-acylation(palmitoylation) of the FIT2 protein promotes its degradation
through a mechanism which involves, but is perhaps not limited
to, the ERAD-C pathway. Although we show that the ERAD-L
pathway is unlikely to be involved, we do not exclude theinvolvement of other protein degradation mechanisms. This link
between SFA-mediated β cell lipotoxicity and FIT2 degradation
impacting LD numbers has not been described previously.

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Further molecular work correlating the degree of FIT2 palmitoylation with its degradation in both physiology and pathology isrequired to further our understanding on how FIT2 function is
ﬁne-tuned at the protein level.
In conclusion, our results describe a means through which
SFAs may disable LD biogenesis in β cells. Palmitoylation of
FIT2 leads to its degradation in part through ERAD-C–mediatedmechanisms. Beyond FIT2 and the understanding of how SFAs
are more damaging than USFAs to cells, our results show that restoration of LD formation, especially in a lipotoxic milieu, such as
obesity and diabetes, is of considerable therapeutic value for preventing β cell dysfunction and loss.
pnas.orgMaterials and Methods
Mice. β cell–speciﬁc FIT2 knockout mice (βFIT2KO, KO) were generated using the
Cre-lox recombination system. Mice with ﬂoxed FIT2 (FIT2ﬂ/ﬂ, FL) (21) were bredwith mice expressing Cre-recombinase, under control of the rat insulin promoter[B6.Cg-Tg(Ins2-cre)25Mgn/J]. Male mice were used in all experiments. βFIT2KO
and their corresponding, ﬂoxed littermates (FIT2ﬂ/ﬂ) were fed with western-type(West) diet (D12079B, Research Diets) from 5 wk of age for a further 25 wk.
B6.BKS (D)-Leprdb/J (db/db) mice were compared with their age-matched, heterozygous littermates (db/+) at 16 wk. Mice were housed in a facility with a 12-h
light–dark cycle and with food and water available ad libitum. All the animal
experiments and protocols were approved by the Institutional Animal Care and
Use Committee of SingHealth (IACUC SingHealth No. 2013/SHS/816) and of
Nanyang Technological University Singapore (A0373).
Islet Isolation, Dispersion, and Cell Culture. Mouse pancreatic islets wereisolated by perfusing the pancreas through the common bile duct with collagenase, as previously described (59). The isolated islets were dispersed into single
cells by incubation with Accutase at 37 ° C for 3 to 5 min. MIN6 cells (passages
30 to 35) were cultured as previously described (60). Transient and stable transfections of MIN6 were performed (SI Appendix). FAs (palmitate, stearate, oleate,
and palmitoleate) were conjugated with FA-free BSA at a 2:1 molar ratio andused at a ﬁnal concentration of 300 μM.

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Immunoblotting and IF Assays. Cells and isolated islets were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor
mixture. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gelelectrophoresis and transferred onto nitrocellulose membranes with blocking,antibody incubation, and image acquisition (SI Appendix). For IF, cryosections of
10 mm thickness from ﬁxed and cryopreserved pancreas were used for IF analysis. Sections were rinsed with Tris-buffered saline (TBS), permeabilized, blockedwith 10% normal goat serum plus 0.2% Triton X-100 in TBS for 1 h at room temperature, and incubated overnight with primary antibodies at 4 ° C in a humidiﬁed atmosphere (SI Appendix).
RNA Extraction, qRT-PCR, RNASeq Library Preparation, and Data Processing. Total RNA was extracted and reverse transcribed according to the manufac-

turer’s instructions (SI Appendix). Real-time PCR was performed on QuantStudio 6
Flex Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR
Master Mix (Applied Biosystems), with intron-spanning primers (Table S1). Pancreatic islets were isolated from mice in quintuplicate. Following overnight recovery,
total RNA was harvested using followed by RNASeq library construction (SIAppendix). Genes were considered to be signiﬁcantly and differentially expressed
when false discovery rate ≤ 0.05, with fragments per kilobase of exon per million
mapped fragments ≥ 1 in one sample group retained for subsequent analysis.
GO-clustering enrichment analysis was carried out on differential genes using the
Functional Annotation tool in DAVID version 6.8 (61, 62), under medium stringency
for all default annotation categories except protein domains. We identiﬁed signiﬁcant, clustered groups having group enrichment scores of ≥1.3, with higher scoresindicative of more signiﬁcant, annotated GO clusters.
Insulin, Intracellular Ca2+, and Membrane Capacitance Measurements.

MIN6 cells and pancreatic islets were preincubated at 37 ° C for 2 h in lowglucose Krebs ringer bicarbonate buffer prior to insulin secretion assay (SIAppendix). To determine islet insulin content, 10 isolated islets were washedtwice with ice-cold phosphate-buffered saline and then lysed with RIPA buffer.
Insulin and protein content of the lysate were measured using mouse insulin
enzyme-linked immunosorbent assay (ELISA) (Mercodia) and bicinchoninic acidassay (Thermo Fisher Scientiﬁc), respectively. Pancreatic insulin content wasdetermined following acid extraction (SI Appendix). Changes in intracellular
Ca2+ and membrane capacitance were recorded from intact pancreatic islets
using fura2-AM on a ﬂuorescence, wide-ﬁeld microscope and a standard, wholecell patch clamp technique, respectively (SI Appendix). For in vivo
glucose–stimulated insulin secretion and ITT, mice were starved for 6 h with ad
libitum access to water and then administered glucose (2 g/kg) or insulin (0.75
unit/kg), respectively. Blood samples were collected from the tail vein prior to,
and 15 min after, intraperitoneal injection. Blood glucose was measured inblood samples collected from tail vein using a glucometer (Accu-Chek Performa
PNAS

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Vol. 119No. 11e2113074119

Nano System). Total blood was centrifuged at 1,000 × g for 10 min, and thesupernatant (serum) was collected for insulin measurements using mouse insulin ELISA (Mercodia).
Ceramides Analysis. Pancreatic islets were prepared (SI Appendix) and ana-lyzed using an Agilent 1290 series Ultra-high-performance liquid chromatographysystem connected to an Agilent 6495 QQQ mass spectrometer after separation
on a ZORBAX Eclipse plus C18 column (2.1 × 50 mm, 1.8 mm, 95 Å, Agilent) at
40 ° C (SI Appendix). Mass spectrometry analysis was performed in positive ion
mode with dynamic, scheduled multiple reaction monitoring (MRM). Mass spectrometry settings, LC-MS gradient, and MRM transitions for each lipid class wereadapted from a previously published method (63). Data analysis was performedon Agilent MassHunter Quantitative analysis software. Relative quantitation was
based on one-point calibration with Ceramide d18:1/8:0. The data were further
normalized to the total number of islets in each sample.
Coimmunoprecipitation, S-acylation, and S-palmitoylation Assays.

MIN6 cells, transiently transfected with pcDNA3.1-FIT2/V5-His, were pretreatedwith 10 mM MG132 (Cell Signaling Technology) for 2 h and thereafter treatedwith 300 mM palmitate (BSA-conjugated) for 6 h. For coimmunoprecipitation, GFPtagged MARCH6 (BC059190) mouse-tagged open reading frame Clone (Origene)
was cotransfected with pcDNA3.1-FIT2/V5-His prior to pretreatment (SI Appendix).
S-palmitoylation assay was performed using CAPTUREome S-Palmitoylated Protein
Kit (Badrilla), in accordance with manufacturer’s instruction (SI Appendix). For ClickiT Palmitoylation assay, MIN6 cells, transiently transfected with a 6HIS-tagged FIT2
overexpression plasmid, were pretreated with 10 mM MG132 (Cell Signaling Technology) for 2 h; then treated with 200 mM Click-IT Palmitic Acid, Azide (Thermo
Fisher Scientiﬁc) for 6 h; and thereafter assay was carried out according to manufacturer’s recommendation without modiﬁcations (SI Appendix).
Statistical Analysis. Data tested for normality (Shapiro–Wilk test) are
expressed as mean 6 SEM. Appropriate statistical analysis using either Student’s
t test, one-way ANOVA with Tukey’s post hoc test, or two-way ANOVA with Sidak
post hoc test were used to determine statistical difference, and P values < 0.05
were considered as statistically signiﬁcant (Prism 7, GraphPad).
Data Availability. RNASeq data reported in this paper are publicly available at
NCBI Gene Expression Omnibus (accession no. GSE133939) (64). Authors
declare no primary datasets and computer codes linked to this study. All otherstudy data are included in the article and/or supporting information.
ACKNOWLEDGMENTS. The mouse pancreatic β cell line MIN6 was kindly
provided by Dr. Jun-ichi Miyazaki, Osaka University, Japan. This work was
supported by the Singapore Ministry of Education grants under its Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2015-T2-2087 and MOE2018-T2-1-085 to Y.A.) and Tier 1 (2017-T1-001-220 and2019-T1-001-059 to Y.A.); the LKCMedicine, Nanyang Technological University Singapore Start-up Grant (P.-O.B.); and the LKCMedicine Healthcare
Research Fund (Diabetes Research), established through the generous support of alumni of Nanyang Technological University, Singapore (Y.A.).
Q.W.C.H. is supported by the Nanyang President’s Graduate Scholarshipaward, Nanyang Technological University, Singapore. J.N.F. is supported bySingapore National Research Foundation Fellowship award (NRF-NRFF201603). Work in the M.R.W. and F.T. laboratories is supported by grants fromthe National University of Singapore via the Life Sciences Institute, theNational Research Foundation (NRF; NRFI2015-05 and NRFSBP-P4) and the
NRF and Agency for Science, Technology and Research (A*STAR) Industry
Alignment Fund–Industry Collaborative Project I1901E0040. N.T. is supported by the 1.3.5 Project for Disciplines of Excellence grant, West China
Hospital, Sichuan University (ZYGD18017). P.-O.B. is supported by the
grants from the Swedish Research Council, the Family Erling-Persson Foundation, the Novo Nordisk Foundation, the Stichting af Jochnick Foundation,
the Swedish Diabetes Association, the Scandia Insurance Company Ltd., theDiabetes Research and Wellness Foundation, Berth von Kantzow’s Foundation, the Strategic Research Program in Diabetes at Karolinska Institutet, theERC-2018-AdG 834860 EYELETS, and the Center of Excellence–International
Collaboration Initiative Grant (139180012, China). X.Z. is currently supported by the National Natural Science Foundation of China grant(82070846). G.A.R. was supported by the Wellcome Trust Investigator

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(WT212625/Z/18/Z) Award, the Medical Research Council Programme grant(MR/R022259/1), and the European Union Horizon2020 research and innovation programme via the Innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement No. 115881 (RHAPSODY). We thank the A*STAR
Microscopy Platform (Singapore) for the electron microscopy support andanalysis.

Author afﬁlitions: aLee Kong Chian School of Medicine, Nanyang Technological
University Singapore, S308232, Singapore; bSingapore Eye Research Institute,Singapore General Hospital, S168751, Singapore; cDepartment of Endocrinology andMetabolism, Center for Diabetes and Metabolism Research, West China Hospital,
Sichuan University, Chengdu 610041, People’s Republic of China; dInstitute of

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Molecular and Cell Biology, Agency for Science, Technology and Research, S138667,
Singapore; eDepartment of Psychological Medicine, Yong Loo Lin School of Medicine,National University of Singapore, S119228, Singapore; fSingapore Lipidomics Incubator,
Department of Medicine, National University of Singapore, S117456, Singapore;gSingapore Lipidomics Incubator, Department of Biochemistry, Life Sciences Instituteand Yong Loo Lin School of Medicine, National University of Singapore, S117456,
Singapore; hHuman Genetics, A*STAR, Genome Institute of Singapore, S138672,
Singapore; iDepartment of Physiology, Yong Loo Lin School of Medicine, National
University of Singapore, S117593, Singapore; jSchool of Biological Sciences, Nanyang
Technological University Singapore, S637551, Singapore; kSection of Cell Biology andFunctional Genomics, Division of Diabetes, Endocrinology, and Metabolism,
Department of Metabolism, Digestion, and Reproduction, Imperial College London,
London SW7 2AZ, United Kingdom; lLe Centre de recherche du Centre hospitalier de de Montre al (CR-CHUM), University of Montre al, Montreal, QC H2X 0A9,
l’Universite
Canada; mSignature Research Program in Cardiovascular and Metabolic Disorders,Duke–National University of Singapore Graduate Medical School, S169857, Singapore;and nThe Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska
Institutet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden

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