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. 2017 Aug 1;26(2):407-418.e3.
doi: 10.1016/j.cmet.2017.07.012.

Triglyceride Synthesis by DGAT1 Protects Adipocytes from Lipid-Induced ER Stress during Lipolysis

Chandramohan Chitraju  1 , Niklas Mejhert  1 , Joel T Haas  2 , L Grisell Diaz-Ramirez  2 , Carrie A Grueter  2 , Jason E Imbriglio  3 , Shirly Pinto  3 , Suneil K Koliwad  4 , Tobias C Walther  5 , Robert V Farese Jr  6
Affiliations

Triglyceride Synthesis by DGAT1 Protects Adipocytes from Lipid-Induced ER Stress during Lipolysis

Chandramohan Chitraju et al. Cell Metab. .

Abstract

Triglyceride (TG) storage in adipose tissue provides the major reservoir for metabolic energy in mammals. During lipolysis, fatty acids (FAs) are hydrolyzed from adipocyte TG stores and transported to other tissues for fuel. For unclear reasons, a large portion of hydrolyzed FAs in adipocytes is re-esterified to TGs in a "futile," ATP-consuming, energy dissipating cycle. Here we show that FA re-esterification during adipocyte lipolysis is mediated by DGAT1, an ER-localized DGAT enzyme. Surprisingly, this re-esterification cycle does not preserve TG mass but instead functions to protect the ER from lipotoxic stress and related consequences, such as adipose tissue inflammation. Our data reveal an important role for DGAT activity and TG synthesis generally in averting ER stress and lipotoxicity, with specifically DGAT1 performing this function during stimulated lipolysis in adipocytes.

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Figures

Figure 1
Figure 1. DGAT1 and DGAT2 in murine WAT are reciprocally regulated in fasting and re-feeding
(A) Relative mRNA levels in gonadal fat, brown fat and livers of ad libitum fed, fasted for 16-h, or after 16-h fasted and 4-h re-fed mice. Cyclophilin was used as reference gene. (B) DGAT1 and DGAT2 enzymatic activities were measured in microsomal fractions of WAT from wild-type mice and DGAT1 KO mice respectively. Data are presented as ± SD. (n=5 mice per group). Statistical significance was evaluated by unpaired two-tailed student’s t test. **p<0.01; ***p<0.001; n.d., not detected.
Figure 2
Figure 2. DGAT1 mediates the majority of TG re-esterification during stimulated lipolysis in vitro
(A) Incorporation of [14C] oleic acid into TG in differentiated 3T3-L1 adipocytes was measured during basal and 3-h stimulated lipolysis by inhibiting DGAT1 (D1i) or DGAT2 (D2i) or both (D1D2i). Lipids were extracted from cells and separated by TLC. Top: Autoradiographs of TLC plates. Lipids from TLC plates were scraped out and quantified by scintillation counter. Data shown represent two independent experiments. (B) FAs release into medium from differentiated 3T3-L1 adipocytes were measured by extracting lipids from the medium and separating them by TLC. FA region on TLC (shown in the graph) was scraped off and radioactivity was measured by liquid scintillation counter. (C) FAs release into medium from differentiated 3T3-L1 adipocytes were measured by counting radioactivity in the medium (In figure 2B, we used a higher quantity of tracer. In subsequent experiments, we reduced tracer amount to label cells). 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<0.01; ***p<0.001. See also Figures S1 and S2.
Figure 3
Figure 3. DGAT1 forms iLDs during stimulated lipolysis
(A) Confocal microscopic images of differentiated 3T3-L1 cells in basal and isoproterenol stimulated lipolysis (ISO). Lipolysis was induced by treating cells with 10 µM isoproterenol in the medium containing no serum and no BSA. LDs were stained with BODIPY 493/503. Scale bar, 10 µm. (B) LDs were quantified using IMARIS software. Representative results from four independent experiments are shown.
Figure 4
Figure 4. DGAT1 mediated re-esterification does not prevent fat loss during fasting or calorie restriction in mice
(A) Western blot analysis showing absence of DGAT1 protein in WAT lysates of ADGAT1 KO mice. (B) DGAT1 activity in WAT microsomal fractions of control and ADGAT1 KO mice. (C) Plasma glucose, glycerol, free FA and TG of 16-h fasted and 4-h re-fed mice (n= 5–6 mice per group). (D) Glycerol release from WAT explants under basal and 3-h stimulated (ISO) lipolysis conditions. (E) Glycerol and FA release from WAT explants treated with DGAT inhibitors under stimulated (ISO) lipolysis for 3-h. (F) Body weights and fat mass loss during 16-h fasting (n= 5–6 mice per group). (G and H) Body weights and change in lean and fat mass during 60% calorie restriction study for 20 days, followed by recovery by ad libitum feeding. (n= 8–10 mice per group). Data are presented as ± SD. Statistical significance was evaluated by unpaired two-tailed student’s t test. *p<0.05; ***p<0.001. See also Figure S3.
Figure 5
Figure 5. DGAT1 inhibition during simulated lipolysis induces the ER stress response in 3T3-L1 adipocytes
(A and B) Heat map and bar graphs showing relative mRNA levels of ER stress marker genes during basal lipolysis determined by RT-qPCR after 10-h treatment with DGAT1 (D1i), DGAT2 (D2i) or both (D1D2i) inhibitors. Thapsigargin (Tg) treated cells were used as positive controls. (C and D) Heat map and bar graphs showing relative mRNA levels of ER stress marker genes during induced lipolysis. Lipolysis was induced by treating cells with 10 µM 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.
Figure 6
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 ad libitum fed, fasted for 16-h, or 6-h cold exposed (while fasting) ADGAT1 KO and control mice. (B) Western blot analysis of Bip and CHOP in WAT of 16-h fasted mice. (C). Relative mRNA levels of inflammatory genes in WAT of ad libitum fed, fasted for 16-h, or 6-h 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.
Figure 7
Figure 7. DGAT1 expression is negatively correlated with ER stress genes in adipose tissue of human subjects
(A) Correlation of DGAT1 or DGAT2 mRNA levels with genes regulated by the unfolded protein response (GO:0006986, n = 167). Results are based on previously published transcriptional profiles generated from human white adipose tissue derived from 26 non-obese and 30 obese women (Arner et al., 2012). Subjects were fasted overnight. Highlighted genes (blue circles) are significantly correlating (p < 0.01) with DGAT1 or DGAT2. (B) Correlation between DGAT1 and a subset of the significant genes from panel (A). Non-obese (n=26) and obese (n=30) subjects are depicted as white and black circles, respectively. Data are expressed as log2 microarray signal.
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