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Highlights
d DGAT-mediated triglyceride synthesis prevents lipid-
induced ER stress
Article
Cell Metabolism 26, 407–418, August 1, 2017 ª 2017 Elsevier Inc. 407
A ad lib 16h fast 4h re-fed B LDs mediates the FA re-esterification cycle in adipocytes is
unclear.
Gonadal fat ** DGAT1
** Here, using a combination of cell and murine models, we
200 investigated how TG synthesis is mediated in adipocytes during
2
** *** lipolysis. We found that the re-esterification cycle is mediated
** ***
exclusively by DGAT1 and that DGAT2 is inactive during these
1 *** 100 ***
*** conditions. Surprisingly, we found that the function of DGAT1-
Activity [A.U]
mediated re-esterification during lipolysis is not to preserve TG
0 0 mass, but instead DGAT1 activation serves to protect the adipo-
Brown fat DGAT2 cyte ER from lipotoxic stress and associated inflammatory con-
Relative mRNA
***
re
h
3 *** gonadal WAT, brown adipose tissue (BAT), and livers from
16
4h
2
P1
AT
pt
AT
EB
D
SR
SO
2i
2i
1D
1D
TG Re-esterification during Stimulated
M
M
1i
2i
1i
2i
A B FA in medium
D
D
D
D
D
210 Lipolysis In Vitro
CpmX104/mg protein
TG (A) Incorporation of [14C] oleic acid into TG in
***
** differentiated 3T3-L1 adipocytes was measured
140 during basal and 3 hr stimulated lipolysis by in-
1,3-DAG FA hibiting DGAT1 (D1i) or DGAT2 (D2i) or both
1,2/2,3- (D1D2i). Lipids were extracted from cells and
DAG Basal Lipolysis Stimulated Lipolysis 70
separated by TLC. (Top) Autoradiographs of TLC
1800 TG plates. Lipids from TLC plates were scraped out
0 and quantified by scintillation counter. Data shown
900 TLC FA represent two independent experiments.
(B) FA release into medium from differentiated 3T3-
DMSO D1i D2i D1D2i
*** *** L1 adipocytes was measured by extracting lipids
0 *** from the medium and separating them by TLC. FA
*** FA C FA in medium
200 region on TLC (shown in the graph) was scraped
200
off, and radioactivity was measured by liquid
CpmX102/mg protein
SO
2i
2i
D 2i
1i
D 2i
1D
1D
D
D
D
D
M
M
D
<1 μm
combinase under the adiponectin pro-
1500 5 moter (Adipoq-Cre; Eguchi et al., 2011).
100 ADGAT1 KO mice lacked detectable
3 DGAT1 protein and had markedly reduced
50
1 DGAT activity in adipose tissue (under con-
0 ditions optimized to detect DGAT1 activity)
but had normal DGAT1 expression in the
ia O
SO
C
C
1i
2i
1i
2i
D al
l
sa
S
D
D
D
D
s
in
in
Ba
M
M
cs
cs
D
ia
and S3A).
Tr
Tr
Decreased Re-esterification in Cultured WAT from Mice DGAT1-Mediated Re-esterification Does Not Preserve
Lacking DGAT1 in Adipocytes Fat Mass during Fasting or Calorie Restriction
Our findings in cultured 3T3-L1 adipocytes show that DGAT1, Identification of DGAT1 as the enzyme responsible for cata-
but not DGAT2, re-esterifies FAs during stimulated lipolysis. To lyzing FA re-esterification during lipolysis in adipocytes, both
Glucose (mg/dl)
DGAT1 120 80 Calorie Restriction in Mice
TG (mg/dl)
90 (A) Western blot analysis showing absence of DGAT1
Tubulin 60
protein in WAT lysates of ADGAT1 KO mice.
60 40
(B) DGAT1 activity in WAT microsomal fractions of
Oo ols
ls O
1 K ontr
ntr 1K 30 20 control and ADGAT1 KO mice.
Co G AT C G AT 0 0
AD AD 16h fast 4h re-fed 16h fast 4h re-fed
(C) Plasma glucose, glycerol, free FA, and TG of 16 hr
fasted and 4 hr re-fed mice (n = 5–6 mice per group).
B DGAT1 activity - WAT (D) Glycerol release from WAT explants under basal
Glycerol (m mol/l)
NEFA (m mol/l) and 3 hr stimulated (ISO) lipolysis conditions.
TG 2.0 0.4
(E) Glycerol and FA release from WAT explants treated
Activity [A. U.]
1.5 0.3 with DGAT inhibitors under stimulated (ISO) lipolysis for
200
1.0 0.2 3 hr.
100 0.5 0.1 (F) Body weights and fat mass loss during 16 hr fasting
*** 0.0 0.0 (n = 5–6 mice per group).
0
16h fast 4h re-fed 16h fast 4h re-fed (G and H) Body weights and change in lean and
Controls ADGAT1 KO
fat mass during 60% calorie restriction study for
D E
Controls ADGAT1 KO Glycerol FFA 20 days, followed by recovery by ad libitum feeding (n =
8–10 mice per group). Data are presented as ±SD.
(n mol/mg protein)
*
(n mol/mg protein)
30
4 and Ron, 2015; Walter and Ron, 2011).
20
Mass (g)
39a
40
1
1
8
90b
24d
pud
jb1
nat
***
frb
1s
1t
em
a5
p
Atf3
Atf4
Dna
Xbp
Xbp
Hsp
2
Pdg
Hgs
Cho
Sec
Relative mRNA
Her
B ip
Pdi
Tm
4
D1i 0 0
D2i 18 Bip *** 120 Atf3 ***
D1D2i 12 *** 60
***
Tg
2 4
-5 -2 0 3 5
0 0
log 2
Tg
SO
Tg
D 2i
2i
D 2i
1i
2i
SO
1i
D
1D
D
D
1D
D
M
M
D
D
C IRE1α/PERK branch targets RIDD D
25 Xbp1s *** DMSO
4 Chop
39a
D1i
1
1
***
90b
24d
pud
jb1
nat
em
D2i
a5
p
***
Atf3
Atf4
Dna
Xbp
Xbp
Hsp
Pdg
Hgs
Cho
Sec
***
Her
Bip
Pdi
Tm
8 2 D1D2i
Relative mRNA
30 min Tg
D1i ** 0
3h 0
10 Bip *** 4 Atf3 ***
***
30 min ***
D2i 4
3h ***
*** 2
2
30 min
D1D2i
3h
0 0
30 min 30 min 3h 30 min 3h
Tg
3h
E Basal Lipolysis Stimulated Lipolysis
-5 -2 0 3 5 Bip
log 2 CHOP
Tubulin
SO 1i 2i 2i Tg SO 1i 2i 2i Tg
M D D 1D M D D 1D
D D D D
Figure 5. DGAT1 Inhibition during Simulated Lipolysis Induces the ER Stress Response in 3T3-L1 Adipocytes
(A and B) Heatmap and bar graphs showing relative mRNA levels of ER stress marker genes during basal lipolysis determined by RT-qPCR after 10 hr treatment
with DGAT1 (D1i), DGAT2 (D2i), or both (D1D2i) inhibitors. Thapsigargin (Tg)-treated cells were used as positive controls.
(C and D) Heatmap and bar graphs showing relative mRNA levels of ER stress marker genes during induced lipolysis. Lipolysis was induced by treating cells with
10 mM isoproterenol in the medium containing no serum and no BSA.
(E) Western blot analysis of Bip and CHOP during basal and induced lipolysis. Data are presented as ±SD (n = 3 biological replicates). Statistical significance
shown is between DMSO control treatments and corresponding inhibitor treatments. Statistical significance was evaluated by unpaired two-tailed Student’s
t test. ***p < 0.001. See also Figures S4 and S5.
2014). Indeed, we found decreased abundance of RIDD target FAs from either de novo synthesis or TG hydrolysis can lead to
mRNAs during prolonged inhibition of both DGAT1 and DGAT2 an ER stress response.
under basal conditions, findings that were also consistent with During lipolysis in adipocytes, our studies show that TG syn-
those after thapsigargin treatment. These data indicate that thesis activity by DGAT2 appears to be inactivated (Figure 2A).
under basal conditions in differentiated adipocytes, either We thus hypothesized that activation of DGAT1 functions during
DGAT1 or DGAT2 activity is sufficient to protect the ER from lipolysis primarily to re-esterify FAs in the ER and prevent disrup-
lipotoxic stress, but in the absence of TG synthesis, the UPR tion of ER function. Indeed, during stimulated lipolysis, inhibition
and ER stress are markedly activated. The increase in ER of DGAT1 alone (or DGAT1 inhibition combined with DGAT2 in-
stress caused by inhibition of both DGAT1 and DGAT2 in the hibition) triggered strong induction of the UPR (16-fold induction
basal conditions was partially blocked by pharmacologic inhi- of Xbp1s after 3 hr of DGAT1 inhibition) and reduced abundance
bition of FA synthesis (data not shown), suggesting that excess of RIDD targets (Figures 5C–5E). These effects were dependent
0 0 0 0
** Il10
Xbp1s 8 F4/80 Ccl2
Relative mRNA
*
6
4
4 ** * 2
4
Relative mRNA
*
2 * 2
0 0 0 0
Bip 10 Cd68 ** Nos2 Arg1
** ** 2
3
3
6
2 * * 1 2
1 * 1
2
0 0 0 0
Atf3 ad lib fasted cold ad lib fasted cold ad lib fasted cold
4
D Controls
2 IL6 ** MCP1 **
19
30 * ADGAT1 KO
Serum Level (pg/ml)
0 20
ad lib fasted cold 3 *
10
0 0
B IL10 TNFα *
Bip * 20
15
*
CHOP 10
10
Tubulin 5
Controls ADGAT1 KO 0 0
fasted cold fasted cold
Figure 6. Lack of DGAT1 in Adipose Tissue Increases the Unfolded Protein Response and Associated Inflammation during Fasting or Cold
Exposure
(A) RT-qPCR analysis of ER stress marker genes in WAT of ADGAT1 KO and control mice fed ad libitum, fasted for 16 hr, or 6 hr cold exposed (while fasting).
(B) Western blot analysis of Bip and CHOP in WAT of 16 hr fasted mice.
(C) Relative mRNA levels of inflammatory genes in WAT of ad libitum fed, fasted for 16 hr, or 6 hr cold-exposed (while fasting) ADGAT1 KO and control mice
determined by RT-qPCR.
(D) Plasma inflammatory markers were estimated by ELISA. Data are presented as ±SD (n = 5–6 mice per group). Statistical significance was evaluated by
unpaired two-tailed Student’s t test. *p < 0.05; **p < 0.01. See also Figure S6.
Second, DGAT1 has broad substrate specificity and can esterify orthologs Are1 and Are2, esterify sterols, a reaction that protects
monacylglycerols, retinol, and long-chain alcohols in addition to cells from accumulation of these lipids. For ACAT enzymes,
DAG (Yen et al., 2005; Yen et al., 2008) and so therefore likely cellular function has been clearly linked to preventing ER stress
protects additionally against toxic accumulation of these lipids due to excess sterols (Devries-Seimon et al., 2005). Maintenance
in the ER. Third, DGAT1 has high activity at high substrate con- of the ER lipid composition and fluidity is likely of crucial impor-
centrations (Cases et al., 2001). Finally, DGAT1 belongs to the tance to cell function, so it is perhaps not surprising that en-
membrane-bound O-acyltransferase (M-BOAT) gene family zymes, such as DGATs and ACATs, monitor and defend against
(Yen et al., 2008), in which several other members function to changes to this composition.
prevent toxic buildup of lipids in the ER. For example, acyl We currently do not know how toxic lipids trigger the UPR
CoA:cholesterol acyltransferase (ACAT) enzymes, or the yeast when DGAT1 activity is absent in adipocytes. The possibility
intestine, allowing organisms to adapt to constantly changing Arner, E., Mejhert, N., Kulyté, A., Balwierz, P.J., Pachkov, M., Cormont, M.,
Lorente-Cebrián, S., Ehrlund, A., Laurencikiene, J., Hedén, P., et al. (2012).
energy conditions.
Adipose tissue microRNAs as regulators of CCL2 production in human
obesity. Diabetes 61, 1986–1993.
STAR+METHODS Bezaire, V., Mairal, A., Ribet, C., Lefort, C., Girousse, A., Jocken, J.,
Laurencikiene, J., Anesia, R., Rodriguez, A.M., Ryden, M., et al. (2009).
Detailed methods are provided in the online version of this paper Contribution of adipose triglyceride lipase and hormone-sensitive lipase to
lipolysis in hMADS adipocytes. J. Biol. Chem. 284, 18282–18291.
and include the following:
Brandt, C., McFie, P.J., and Stone, S.J. (2016). Diacylglycerol acyltransfer-
d KEY RESOURCES TABLE ase-2 and monoacylglycerol acyltransferase-2 are ubiquitinated proteins
d CONTACT FOR REAGENT AND RESOURCE SHARING that are degraded by the 26S proteasome. Biochem. J. 473, 3621–3637.
d METHOD DETAILS Buhman, K.K., Smith, S.J., Stone, S.J., Repa, J.J., Wong, J.S., Knapp, F.F., Jr.,
Burri, B.J., Hamilton, R.L., Abumrad, N.A., and Farese, R.V., Jr. (2002). DGAT1
B Animals and Dietary Intervention Studies
is not essential for intestinal triacylglycerol absorption or chylomicron synthe-
B 3T3-L1 Cell Differentiation
sis. J. Biol. Chem. 277, 25474–25479.
B Culture and Differentiation of Human Multipotent Adi-
Cases, S., Smith, S.J., Zheng, Y.W., Myers, H.M., Lear, S.R., Sande, E., Novak,
pose-derived Stem Cells S., Collins, C., Welch, C.B., Lusis, A.J., et al. (1998). Identification of a gene
B [14C]- Oleic Acid Labeling of Lipids, Lipid Extraction encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacyl-
and Thin Layer Chromatography glycerol synthesis. Proc. Natl. Acad. Sci. USA 95, 13018–13023.
B DGAT Activity Assay Cases, S., Stone, S.J., Zhou, P., Yen, E., Tow, B., Lardizabal, K.D., Voelker, T.,
B Ex Vivo Lipolysis Assay and Farese, R.V., Jr. (2001). Cloning of DGAT2, a second mammalian diacyl-
B Microscopy and Image Processing glycerol acyltransferase, and related family members. J. Biol. Chem. 276,
38870–38876.
B RNA Extraction and Quantitative Real-Time PCR
(qRT-PCR) Chen, H.C., Smith, S.J., Ladha, Z., Jensen, D.R., Ferreira, L.D., Pulawa, L.K.,
McGuire, J.G., Pitas, R.E., Eckel, R.H., and Farese, R.V., Jr. (2002a). Increased
B Immunoblotting
insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltrans-
B DGAT Inhibitors
ferase 1. J. Clin. Invest. 109, 1049–1055.
d QUANTIFICATION AND STATISTICAL ANALYSIS
Chen, H.C., Stone, S.J., Zhou, P., Buhman, K.K., and Farese, R.V., Jr. (2002b).
Dissociation of obesity and impaired glucose disposal in mice overexpressing
SUPPLEMENTAL INFORMATION acyl coenzyme a:diacylglycerol acyltransferase 1 in white adipose tissue.
Diabetes 51, 3189–3195.
Supplemental Information includes seven figures and one table and can be Choi, K., Kim, H., Kang, H., Lee, S.Y., Lee, S.J., Back, S.H., Lee, S.H., Kim,
found with this article at http://dx.doi.org/10.1016/j.cmet.2017.07.012. M.S., Lee, J.E., Park, J.Y., et al. (2014). Regulation of diacylglycerol
Villanueva, C.J., Monetti, M., Shih, M., Zhou, P., Watkins, S.M., Bhanot, S., and Zechner, R., Zimmermann, R., Eichmann, T.O., Kohlwein, S.D., Haemmerle,
Farese, R.V., Jr. (2009). Specific role for acyl CoA:Diacylglycerol acyltransfer- G., Lass, A., and Madeo, F. (2012). FAT SIGNALS—lipases and lipolysis in lipid
ase 1 (Dgat1) in hepatic steatosis due to exogenous fatty acids. Hepatology metabolism and signaling. Cell Metab. 15, 279–291.
50, 434–442.
Zhao, T.J., Liang, G., Li, R.L., Xie, X., Sleeman, M.W., Murphy, A.J.,
Volmer, R., and Ron, D. (2015). Lipid-dependent regulation of the unfolded Valenzuela, D.M., Yancopoulos, G.D., Goldstein, J.L., and Brown, M.S.
protein response. Curr. Opin. Cell Biol. 33, 67–73. (2010). Ghrelin O-acyltransferase (GOAT) is essential for growth hormone-
Volmer, R., van der Ploeg, K., and Ron, D. (2013). Membrane lipid saturation mediated survival of calorie-restricted mice. Proc. Natl. Acad. Sci. USA 107,
activates endoplasmic reticulum unfolded protein response transducers 7467–7472.
Further information and request for reagent and resources should be directed to Robert V. Farese, Jr. (robert@hsph.harvard.edu) or
Tobias C. Walther (twalther@hsph.harvard.edu).
METHOD DETAILS
[14C]- Oleic Acid Labeling of Lipids, Lipid Extraction and Thin Layer Chromatography
DGAT1 and DGAT2 specific activities in differentiated 3T3-L1 adipocytes were determined by [14C]-oleic acid incorporation into TG
in presence of inhibitors. Cells were pre-treated for 30 min with 5 mM inhibitors, cells were pulse labeled with [14C]-oleic acid
(50 mCi/mmol) for 3 hr in presence of inhibitors. To determine activities during induced lipolysis, cells were treated with 10 mM isopro-
terenol. Cells were washed with phosphate buffer saline for 3 times. Lipids were extracted directly from 6-well cell-culture plates by
adding hexane: isopropanol mixture (3:2) and gentle shaking for 10 min. The process is repeated second time for efficient extraction
of all lipids. Lipids were dried under nitrogen stream and separated by TLC using hexane:diethyl ether:acetic acid (80:20:1) solvent
system. TLC plate were exposed to phosphor imaging cassette overnight and revealed by Typhoon FLA 7000 phosphor imager.
Lipids on TLC plate were stained with iodine vapors; bands were scraped and quantified by liquid scintillation counter. After lipid
extraction from 6-well plates, 400 mL of lysis buffer (0.3 N NaOH and 0.1% SDS) was added to each well and kept for shaking for
3 hr to lyse the cells for protein measurement by Bio-Rad DC Protein Assay kit.
Immunoblotting
Cells were lysed using RIPA lysis buffer. Tissues were lysed in buffer containing 250 mM sucrose, 100 mM Tris-HCl (pH 7.4), and
protease inhibitors in a dounce homogenizer. Proteins were denatured in Laemmli buffer and were separated on 10% SDS-PAGE
gel and transferred to PVDF membrane (Bio-Rad). The membranes were blocked with blocking buffer for 2 hr in TBST containing
5% BSA or 5% milk, and then incubated with primary antibodies overnight. The membranes were then washed with TBST for
10 min x 3 times, and incubated in mouse secondary antibodies (Santa Cruz Biotechnology) at 1:5000 dilutions in blocking buffer.
Membranes was washed again with TBST for 10 min x 3 times and revealed using the Super Signal West Pico kit (Thermo Scientific).
DGAT1 antibodies were a gift from Dr. Jin Ye from UT Southwestern Medical Center. CHOP and Bip antibodies were purchased from
Cell Signaling Technology.
DGAT Inhibitors
DGAT2 and DGAT1 inhibitors were obtained from collaborators at Merck & Co., Inc. DGAT2 and DGAT1 inhibitors are well charac-
terized and published. (Imbriglio et al., 2015; Liu et al., 2013). Inhibitors were dissolved in DMSO. For [14C]-oleic acid labeling studies,
Cells were pre-treated for 30 min with 5 mM inhibitors, cells were pulse labeled with [14C]-oleic acid (50 mCi/mmol) for 3 hr in presence
of inhibitors.
Data are presented as means ± SD. Statistical significance was evaluated by unpaired two-tailed Student’s t test using Microsoft
Excel software.