XBP1s Is an Anti-lipogenic Protein*□S

Received for publication, March 24, 2016, and in revised form, June 12, 2016 Published, JBC Papers in Press, June 20, 2016, DOI 10.1074/jbc.M116.728949

Hilde Herrema‡1, Yingjiang Zhou‡, Dongyan Zhang§, Justin Lee‡, Mario Andres Salazar Hernandez‡,Gerald I. Shulman§¶, and Umut Ozcan‡2

From the ‡Division of Endocrinology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, the§Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06519, and Departments of¶Internal Medicine and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

Endoplasmic reticulum (ER) stress has been shown to contrib-ute to various metabolic diseases, including non-alcoholic fattyliver disease and type 2 diabetes. Reduction of ER stress by treat-ment with chemical chaperones or overexpression of ER chap-erone proteins alleviates hepatic steatosis. Nonetheless, X-boxbinding protein 1s (XBP1s), a key transcription factor thatreduces ER stress, has been proposed as a lipogenic transcrip-tion factor. In this report, we document that XBP1s leads tosuppression of lipogenic gene expression and reduction ofhepatic triglyceride and diacylglycerol content in livers of diet-induced obese and genetically obese and insulin-resistant ob/obmice. Furthermore, we also show that PKC� activity, which cor-relates with fatty liver and which causes insulin resistance, wassignificantly reduced in diet-induced obese mice. Finally, wehave shown that XBP1s reduces the hepatic fatty acid synthesisrate and enhances macrolipophagy, an initiating step in lipoly-sis. Our results reveal that XBP1s reduces hepatic lipogenic geneexpression and improves hepatosteatosis in mouse models ofobesity and insulin resistance, which leads us to conclude thatXBP1s has anti-lipogenic properties in the liver.

The increasing worldwide prevalence of obesity is consid-ered one of the most serious public health problems of the 21stcentury (1, 2). Obesity is a predisposing factor for various met-abolic disorders, including type 2 diabetes, cardiovascular dis-ease, and non-alcoholic fatty liver disease (NAFLD)3 (3, 4). Asubset of NAFLD patients develops non-alcoholic steatohepa-titis, liver cirrhosis, and liver failure and eventually requires aliver transplantation (5–7). Additionally, NAFLD is highly asso-ciated with development of insulin resistance, and the vastmajority of obese and type 2 diabetic patients are diagnosed

with NAFLD (8, 9). Despite extensive efforts in the field, theunderlying molecular mechanisms for NAFLD in obesity arepoorly understood.

Endoplasmic reticulum (ER) stress, a condition in which ERhomeostasis is disturbed by pathologies that include, but arenot limited to, accumulation of misfolded proteins and overtnutrient supply, has been identified as an important player inthe pathogenesis of metabolic diseases such as obesity, insulinresistance, and type 2 diabetes (10). ER stress leads to activationof the unfolded protein response (UPR), a complex signalingnetwork that aims to resolve ER stress and restore ER homeo-stasis (11, 12). Inositol-requiring enzyme 1 (IRE1) is one of thethree master modulators of UPR signaling. Together with PKR-like endoplasmic reticulum kinase and activating transcriptionfactor 6 (ATF6), IRE1 senses ER luminal status and, accord-ingly, activates one of the most crucial signaling arms of theUPR. Activated IRE1 cleaves X-box binding protein 1 (XBP1)mRNA, leading to the formation of the spliced form of XBP1(XBP1s), a highly active transcription factor and central regu-lator of ER homeostasis.

We have shown previously that nuclear translocation ofXBP1s is severely impaired in the liver of obese and insulin-resistant mice (13). Reinstating the hepatic activity of XBP1sreduced ER stress and improved glucose homeostasis throughmechanisms that are both dependent (14, 15) and independentof XBP1s transcriptional activity (16). This indicates thatXBP1s is indispensable for the maintenance of glucose homeo-stasis in obesity. Indeed, XBP1 haplodeficiency led to the devel-opment of glucose intolerance and insulin resistance inHFD-fed BALB/c mice, a strain that is otherwise resistant todiet-induced insulin resistance (17).

Fatty livers of both obese rodents (17) and humans (18) haveincreased levels of ER stress. Importantly, alleviation of ERstress by chemical chaperone treatment (19) or overexpressionof ER chaperone genes (20) reverses hepatic steatosis. Further-more, pharmacological activation of ER stress results in hepaticsteatosis in mice deficient in ER stress-sensing or ER qualitycontrol pathways (21, 22). Liver-specific IRE1�-null mice,which are deficient in XBP1s activity, have enhanced develop-ment of fatty liver upon pharmacological induction of ER stress(23). This suggests that XBP1s potentially serves a protectiverole in the development of hepatic steatosis. In contrast to thesereports, which all indicate that increased ER stress promoteshepatic steatosis, it has been suggested that XBP1s, which isknown to reduce ER stress and increase ER adaptive capacity, isa lipogenic transcription factor (24). Considering this discrep-

* This work was supported by funds from the Department of Medicine, Bos-ton Children’s Hospital (to U. O.); National Institutes of Health GrantsR01DK081009 and R01DK098496 (to U. O.), R01 DK-40936, and U24DK-059635; and by American Diabetes Association Career DevelopmentGrant 7-09-CD-10. U. O. is a scientific founder, shareholder, and member ofthe scientific advisory board and board of directors of ERX Pharmaceuti-cals, Inc. The content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institutes of Health.

□S This article contains supplemental Figures S1–S4.1 To whom correspondence may be addressed. E-mail: hilde.herrema@

childrens.harvard.edu.2 To whom correspondence may be addressed. E-mail: umut.ozcan@

childrens.harvard.edu.3 The abbreviations used are: NAFLD, non-alcoholic fatty liver disease; ER,

endoplasmic reticulum; UPR, unfolded protein response; HFD, high-fatdiet; DIO, diet-induced obese; GTT, glucose tolerance test; ITT, insulin tol-erance test; TG, triglyceride(s); DAG, diacylglycerol; ADRP, adipose differ-entiation-related protein.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 33, pp. 17394 –17404, August 12, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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ancy and also taking into account that XBP1s activity is reducedin mouse models of obesity and NAFLD (13), we sought toinvestigate the role of XBP1s in the development of NAFLD.

Results

XBP1s Reduces Hepatosteatosis in DIO Mice—To investigatethe effect of XBP1s on hepatic steatosis, first C57Bl/6J micewere fed an HFD (45% kcal) for 12 weeks to induce obesity,insulin resistance, and NAFLD. Mice were then injectedthrough the tail vein with a replication-incompetent adenovi-

rus expressing XBP1s (adXBP1s) or LacZ (adLacZ) as a control.In line with our previous observations, 6-h fasted blood glucoselevels were significantly reduced in adXBP1s-injected DIOmice compared with ad-LacZ injected controls (Fig. 1A). Wenext preformed a glucose tolerance test (GTT) by intraperito-neal injection of glucose on post-injection day 5. Glucose toler-ance was significantly improved in adXBP1s-injected micecompared with the controls (Fig. 1B). Additionally, insulin-me-diated reduction in blood glucose levels, as determined by insu-lin tolerance test (ITT) 7 days post-injection, was significantly

FIGURE 1. XBP1s reduces hepatic triglyceride content and lipogenic gene expression. C57BL6/J mice (3 weeks old) were fed an HFD (45% kcal) for 12 weeksand injected with adLacZ, adXBP1s, or �DBD-XBP1s (8*10E7 pfu/g) through the tail vein. A, blood glucose levels were assessed after 6 h of fasting onpost-adenovirus injection day 9. B, a GTT with 1 g/kg glucose (i.p. injection) was performed on day 5 post-adenovirus injection. C, an ITT was performed on day7 post-adenovirus injection by intraperitoneal insulin (0.75 IU/kg) injection. D, plasma insulin levels after 6-h fasting on day 9 post-adenovirus injection. E,homeostatic model assessment for insulin resistance as calculated from 6 h-fasted glucose levels and corresponding plasma insulin levels on day 9 post-adenovirus injection. F, average daily food intake (g/mouse/day) and G, body weight (g). H, relative hepatic mRNA levels of Xbp1s, Srebp1c, FasN, Acc1, Acc2, andScd1 in livers of adLacZ and adXBP1s-injected mice. Gene expression was normalized using the 18s gene as a housekeeping gene. I and J, liver triglyceridecontent (I) and plasma triglyceride levels (J) were determined in 6 h-fasted mice on post-injection day 9. K, immunoblotting of XBP1s and FoxO1 in the nucleusand FasN in total homogenates of livers of LacZ and XBP1s-expressing mice. NUP98 was used as a loading control for nuclear lysates, whereas Tubulin servedas a loading control for total liver lysates. Error bars are represented as mean � S.E., and p values were determined by Student’s t test or two-way analysis ofvariance testing with Bonferroni post-test for multiple comparison analysis (for GTT and ITT). *, p � 0.05; **, p � 0.01; ***, p � 0.001.

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improved in XBP1s-injected mice (Fig. 1C). Circulating plasmainsulin levels were significantly lower than those of the controlgroup (Fig. 1D), and homeostatic model assessment for insulinresistance was reduced by �60% (Fig. 1E) in adXBP1s-injectedmice compared with the controls. Notably, these beneficialchanges in glucose homeostasis were established independentof changes in food intake and body weight (Fig. 1, F and G).These results indicate that, in addition to the genetically obeseand diabetic ob/ob mice (13–16), reinstatement of XBP1saction in the liver of DIO mice significantly improves glucosehomeostasis.

We then assessed the parameters of hepatic lipid metabolismin XBP1s-overexpressing mice versus the controls. Surpris-ingly, expression of key lipogenic genes, including sterol regu-latory element-binding protein 1c (Srebp1c), fatty acid synthase(FasN), and stearoyl-CoA desaturase 1 (Scd1), were signifi-cantly reduced in the livers of adXBP1s-injected mice com-pared with the adLacZ-injected controls (Fig. 1H). Quantitativeanalysis revealed a 30% reduction in TG content in livers ofXBP1s-injected mice compared with LacZ-injected controls(Fig. 1I). XBP1s expression, however, led to a significantincrease in circulating TG levels (Fig. 1J), probably because ofresolution of lipids from the liver to the circulation. Plasma andhepatic cholesterol levels were unaffected by XBP1s expression(Fig. S1, A and B). Protein levels of fatty acid synthase, a centralenzyme in de novo lipogenesis, were significantly reduced (Fig.1K). In line with our previous report in ob/ob mice (16), XBP1sexpression led to exclusion of FoxO1 from the nucleus in theDIO model too (Fig. 1K).

XBP1s Reduces Hepatic Diacylglycerol Levels and InhibitsPKC� Translocation—TG in the liver are primarily formedthrough esterification of fatty acids (25). Diacylglycerol (DAG),which consists of a glycerol backbone with two fatty acylgroups, is an intermediate of the TG synthetic pathway and aTG precursor molecule. In addition, DAG is a potent secondmessenger signaling molecule with the potential to activatePKC family members (26). Although, in obesity and NAFLD,many lipid species accumulate ectopically (e.g. in the liver),there has been specific attention on hepatic DAG accumulationand intracellular DAG localization following reports that DAGscan directly facilitate insulin resistance in NAFLD by activatingthe novel PKC isoform � (PKC�) (27–29). DAG-activated PKC�subsequently binds to the insulin receptor, thereby preventingdownstream insulin-mediated signaling. Among the PKC iso-forms, PKC� has been shown to be highly increased in NAFLDand to correlate strongly with hepatic DAG content both inrodents (26, 28) and humans (30). Furthermore, intracellularDAG levels and subsequent activation of PKC� have been putforward as reliable predictors of insulin resistance in fatty liversof obese patients (30, 31).

Considering that TG content was significantly reduced in thelivers of adXBP1s-injected DIO mice, we investigated whetherXBP1s could also reduce hepatic DAG levels. For this purpose,we first analyzed total DAG levels in the livers of adXBP1s- andadLacZ-injected DIO mice. We found that XBP1s expressionled to a slight but not significant (p � 0.099) decrease in totalhepatic DAG levels (Fig. 2A). However, when the DAG contentof hepatic cytosolic and membrane compartments was ana-

lyzed, we observed a significant reduction in DAG levels in thecytosolic compartment of the livers of XBP1s-injected mice(Fig. 2A).

Although gene expression levels of Pkrce in livers ofadXBP1s-injected mice were reduced compared with controls(Fig. 2B), total PKC� protein levels remained unaffected (Fig.2C). Cellular localization of PKC�, which can be analyzed by theratio of cytosolic to membrane PKC�, is an important determi-nant for PKC� activity (32, 33). Thus, we next analyzed PKC�levels in cytosolic and membrane compartments of livers ofadXBP1s- and adLacZ-injected mice. Although PKC� levelswere similar in cytosolic compartments of livers of adXBP1s-injected mice and their controls (Fig. 2D), the membrane con-tent was significantly reduced in adXBP1s-injected mice (Fig.2D). Therefore, the ratio of membrane/cytosolic PKC� was sig-nificantly reduced in adXBP1s-injected mice (Fig. 2E), indicat-ing a decrease in PKC� activity.

Next we analyzed the levels of 13 different DAG species bothin the cytosolic and membrane compartments by usingLC/MS/MS in adLacZ- and adXBP1s-injected DIO mice. 10 of13 analyzed DAG species were decreased in the cytosolic frac-tion of livers of XBP1s-expressing mice (Fig. 2F). 4 of 13 ana-lyzed membrane DAG species were moderately but signifi-cantly increased in livers of XBP1s-expressing mice (Fig. 2G).Total ceramide levels remained unaffected; however, weobserved differential regulation of various ceramide species inlivers of XBP1s-expressing mice (Fig. S2, A and B).

These results establish a novel role for XBP1s in the regula-tion of hepatic DAG levels and PKC� activation. In addition, theresults are suggestive of a regulatory role of XBP1s in glucosehomeostasis through inhibition of PKC� activity as a result ofreduced DAG content.

XBP1s Reduces the Hepatic Fatty Acid Synthesis Rate andIncreases Macrolipophagy in ob/ob Mice—We next investi-gated whether the effects of XBP1s on the lipogenic geneexpression profile seen in the DIO mouse model could bereproduced in obese and insulin-resistant ob/ob mice, whichhave severe accumulation of lipids in the liver. For this purpose,we injected adXBP1s or adLacZ into the tail vein of 10-week-old ob/ob mice. On post-injection day 9, we sacrificed the miceand extracted the livers for further investigation. QuantitativePCR analysis revealed significant reductions in the expres-sion of lipogenic genes such as Acc1, Acc2, and FasN (Scd1levels were reduced but without reaching statistical differ-ence) in XBP1s-expressing ob/ob mice compared with thecontrols (Fig. 3A).

The above findings indicate that XBP1s is able to reduce lipo-genic gene expression in a diet-induced and a genetic model ofobesity and insulin resistance in mice. Most of the effects ofXBP1s on metabolic homeostasis have been attributed to theability of XBP1s to act as a transcription factor by directly bind-ing to the DNA, thereby regulating gene expression. To assesswhether the DNA-binding capacity of XBP1s was a prerequisiteto mediate the reduction in lipogenic gene expression, wetested whether a DNA-binding-defective mutant XBP1s(�DBD-XBP1s) could recapitulate the effects on lipogenic geneexpression. Using an adenovirus-mediated approach, weexpressed LacZ, wild-type XBP1s, and �DBD-XBP1s, in which

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the DNA-binding domain was replaced with an artificialnuclear localization signal (16), in the liver of ob/ob mice. Asanticipated, expression of wild-type XBP1s led to a significant

induction of XBP1s-mediated UPR genes such as Erdj4, Grp58,and Grp78 (Fig. S3). However, as we reported previously,�DBD-XBP1s was unable to increase expression of these genes.

FIGURE 2. XBP1s reduces hepatic diacylglycerol content and PKC� activity. C57Bl6/J mice were fed an HFD (45% kcal) for 12 weeks (starting at 3 weeks ofage) and injected with adLacZ or adXBP1s (8*10E7 pfu/g) through the tail vein. A, DAG content was determined in in cytosolic (black columns) and membrane(gray columns) compartments in livers of 6 h-fasted mice on post-injection day 9. Total DAG content was calculated from the sum of cytosolic and membraneDAG content. B, relative mRNA levels of Pkrce. Gene expression was normalized using the 18s gene as a housekeeping gene. C, Western blotting analysis of PKC�protein in whole liver homogenates. Tubulin was used as a loading control. D, PKC� protein levels were determined in the cytosol (top panel) and membrane(bottom panel) compartments of livers of 6 h-fasted mice 9 days post-injection. GAPDH was used as a loading control for cytosolic lysate, whereas NaATPaseserved as a loading control for the membrane lysates. E, PKC� protein expression was quantified using densitometric analysis of the autoradiographical signalof PKC� in the membrane and cytosol. The membrane-to-cytosol (M/C) ratio as measure of PKC� activity is expressed in the graph. Hepatic DAG species wereanalyzed in the cytosol (F) and membrane (G) compartments of livers of adLacZ- and adXBP1s-expressing mice. Error bars are represented as mean � S.E.; pvalues were determined by Student’s t test. *, p � 0.05; **, p � 0.01; ***, p � 0.001; ns, not significant.

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In contrast to the chaperone expression profile, expression oflipogenic genes was reduced to the same extent in adXBP1s-and �DBD-expressing mice. This implies that the XBP1s-me-diated reduction in lipogenic gene expression is independent ofits DNA-binding capacity. We have shown previously thatXBP1s, independent of its DNA-binding capacity, can directlyinteract with FoxO1 and promote its degradation in the 26Sproteasome (16). We therefore postulate that the effect ofXBP1s on lipogenic gene expression probably relies on directinteraction of XBP1s with lipogenic gene regulatory proteinand its inhibition.

To further investigate the hepatic lipid phenotype in miceexpressing adXBP1s, we next performed H&E staining to deter-mine the histopathological changes in the livers. As shown inFig. 3B, H&E staining documented that XBP1s reduced thenumber and size of the lipid droplets in the livers of XBP1s-expressing ob/ob mice compared with the controls. Quantita-tive analysis of hepatic TG content revealed a close to signifi-cant reduction in the livers of ob/ob mice expressing XBP1s(Fig. 3C).

Next we performed an in vivo lipogenesis assay to assess thehepatic fatty acid synthesis rate. For this, adXBP1s- and

FIGURE 3. Enhanced macrolipophagy in XBP1s-injected ob/ob mice. Leptin-deficient obese and diabetic ob/ob mice (10 weeks old) on a C57Bl6/J back-ground were injected with adLacZ or adXBP1s (8*10E7 pfu/g) through the tail vein. A, relative hepatic mRNA levels of the lipogenic genes Acc1, Acc2, FasN, andScd1 in livers of adLacZ- and adXBP1s-injected mice. Gene expression was normalized using the 18s gene as a housekeeping gene. B, livers were fixed andprepared for H&E staining. Pictures are representative sections of livers of adLacZ- and adXBP1s-injected mice (�4 magnification). C, liver triglyceride contentwas determined in 6 h-fasted mice on day 9 post-adenovirus injection. D, the hepatic fatty acid synthesis rate was determined by [3H2O] (2 mCi/mouse)injection on day 5 post-injection. Fatty acids were extracted and subsequently analyzed by TLC. The rate of hepatic fatty acids was calculated as the amount of[3H] incorporated into fatty acids per gram of tissue per hour. E, plasma triglyceride levels were determined in 6 h-fasted mice on day 9 post-adenovirusinjection. F, livers of 6 h-fasted mice were fixed for electron microscopy analysis on day 9 post-adenovirus injection. Pictures are representative electronmicroscopic images of lipid droplets in the liver of adXBP1s and adLacZ-injected mice. G, Western blotting analysis of ATG5 protein in whole liver homogenates.Tubulin was used as a loading control (top panel). ATG5 and tubulin protein expression levels were quantified using densitometric analysis and expressed asa ratio ATG5/tubulin in adLacZ and adXBP1s-injected mice (bottom panel). H, Western blotting analysis of LC3IIB and ADRP (as a control) protein levels inisolated lipid droplets from livers of adLacZ and adXBP1s-expressing mice. Note that each lane contains proteins isolated from lipid droplets of one ob/obmouse liver (top panel). LC3BII and ADRP protein expression levels were quantified using densitometric analysis and expressed as a ratio of LC3BII/ADRP inadLacZ- and adXBP1s-injected mice (bottom panel). Error bars are represented as mean � S.E.; p values were determined by Student’s t test. *, p � 0.05; **, p �0.01; ***, p � 0.001.

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adLacZ-injected mice were injected i.p. with tritiated water(3H2O) as a chaser on post-injection day 5. Livers were excisedfor analysis of newly synthesized fatty acids 1 h after the injec-tion. The results indicated that the hepatic fatty acid synthesisrate in adXBP1s-injected mice was significantly (40%) lowercompared with the controls (Fig. 3D). XBP1s expression alsoincreased plasma TG levels in ob/ob mice (Fig. 3E).

Based on the suggestive reduction in lipid droplet sizeobserved in H&E-stained livers of XBP1s-injected mice, wedecided to further investigate the lipid droplet morphology.Using an electron microscopy approach, we observed increaseddouble membrane structures in the lipid droplets that weresuggestive of autophagosome formation in adXBP1s-injectedob/ob mice compared with those in the control group (Fig. 3F).Degradation of lipid droplets through autophagy (also termedmacrolipophagy) has been suggested as an important process inregulation of hepatic lipid metabolism (34). To investigatewhether macrolipophagy was indeed increased in the livers ofXBP1s-expressing mice, we first determined hepatic proteinlevels of autophagy gene 5 (ATG5), a protein with a crucial rolein early-stage autophagosome formation and thus in lipid drop-let breakdown. ATG5 levels were markedly increased in thelivers of ob/ob mice expressing XBP1s compared with LacZcontrols, which indicates increased autophagosome formation(Fig. 3G). During autophagy, the cytosolic form of microtubule-associated protein 1A/1B light chain 3 (LC3BI) is conjugated tophosphatidylethanolamine to form LC3BII, which is subse-quently recruited to autophagosomal membranes. LC3BII sub-sequently mediates the degradation of autophagosomal con-tent. To determine the level of LC3BII associated with lipiddroplets, we isolated hepatic lipid droplets and analyzed LC3BIIlevels in this compartment. Lipid droplet-associated LC3BIIwas significantly increased in the livers of adXBP1s-injectedob/ob mice (Fig. 3H), which indicates enhanced autophago-some formation. These results suggest that XBP1s, in additionto reducing the hepatic lipogenic rate, enhances breakdown ofthe lipid droplets by promoting autophagy.

XBP1s Reduces Hepatic Lipogenic Gene Expression in Fruc-tose-fed Mice—Our results indicate that XBP1s lowers hepaticlipogenic gene expression in DIO and ob/ob mice and exertsanti-lipogenic effects. These results contradict the conclusionsfrom a previous study (24), which concluded that XBP1sincreases lipogenesis in mice fed a highly lipogenic fructosediet. To assess the effect of XBP1s expression in a setting with ahigh lipogenic rate without the confounding effects of obesityand insulin resistance, we fed mice a 60% fructose diet for 4weeks. As expected, high-fructose feeding significantly inducedexpression of lipogenic genes (Fig. S4A). Nevertheless, we didnot observe the previously reported increase in Xbp1s geneexpression levels in fructose-fed mice compared with chow-fedmice (24). After 4 weeks of fructose feeding, mice were injectedwith adXBP1s or adLacZ. XBP1s expression significantlyreduced expression of the key lipogenic genes Srebp1c, FasN,and Scd1 and led to a non-significant reduction in Acc1 andAcc2 (Fig. 4A). However, in contrast to DIO and ob/ob mousemodels, XBP1s overexpression in this setting did not reducehepatic TG content (Fig. 4B). Notably, we again observedincreased plasma TG levels in XBP1s-expressing, fructose-fed

FIGURE 4. XBP1s reduces lipogenic gene expression in livers of fructose-fed mice. C57Bl6/J mice were fed a fructose diet for 4 weeks (starting at 8weeks of age) and then injected with adLacZ or adXBP1s (8*10E7 pfu/g)through the tail vein. A, relative hepatic mRNA levels of Xbp1s, Srebp1c, FasN,Acc1, Acc2, and Scd1 livers of adLacZ- and adXBP1s-injected mice on day 9post-adenovirus injection. Gene expression was normalized using the 18sgene as a housekeeping gene. B and C, liver triglyceride content (B) andplasma triglyceride levels (C) were determined in 6 h-fasted mice on day 9post-adenovirus injection. D, blood glucose levels as determined in 6 h-fastedmice on day 9 post-adenovirus injection. E and F, average daily food intake (E)and body weight (F) in 6 h-fasted mice on day 9 post-adenovirus injection.Error bars are represented as mean � S.E.; p values were determined byStudent’s t test. *, p � 0.05; **, p � 0.01; ***, p � 0.001.

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mice compared with the controls (Fig. 4C). Additionally,XBP1s-expressing mice had a significant reduction in bloodglucose levels in this setting too (Fig. 4D). These alterationswere achieved without changes in food intake or body weight(Fig. 4, E and F). Liver and plasma hepatic cholesterol levelswere unaffected by XBP1s expression in high fructose-fed mice(Fig. S4, B and C).

Discussion

NAFLD is one of the most serious pathologies of obesity, andit is currently the most common liver disease worldwide (35,36). There is a strong association between obesity and NAFLD,and around 90% of obese, type 2 diabetic patients are diagnosedwith NAFLD (37). NAFLD and type 2 diabetes increase mor-bidity and mortality rates in the obese population and create aserious economic burden on affected societies (38 – 40).NAFLD significantly increases the incidence of liver cirrhosisand hepatocellular carcinoma in the obese population (41).Despite continuing efforts in the field, the available treatmentregimens can only minimally reduce NAFLD, and better treat-ment modalities have not been developed because of the factthat the underlying molecular pathologies are poorly under-stood. Identifying the molecular mechanisms that lead toNAFLD is therefore of great importance.

We (14 –16) and others (42, 43) have previously identifiedXBP1s as a crucial regulator of glucose homeostasis in obesity.XBP1s action is severely impaired in mouse models of obesity(13), and this relative “XBP1s deficiency” contributes to thedevelopment of sustained ER stress, pathological activation ofUPR signaling (13), and, ultimately, to disturbed glucose home-ostasis. Reducing ER stress with chemical chaperones in vivo orby reinstating XBP1s action reduces ER stress and improvesglucose intolerance and insulin resistance (14, 15, 19, 20).

Increased ER stress in obesity has also been implicated in thedevelopment of hepatosteatosis (21, 22). Despite several previ-ous reports indicating that a homeostatic ER condition is criti-cal for the maintenance of hepatic lipid homeostasis in obesity(19 –23) and that reduction of ER stress is an anti-lipogenicmodification, XBP1s, which probably has the strongest capacityto reduce ER stress, was suggested as a lipogenic factor (24).These postulates created a serious dilemma in the field andscrutinized the possibility of development of therapeuticapproaches to reactivate XBP1s in the liver to treat obesity-associated insulin resistance and type 2 diabetes.

Here we document that, contrary to previous suggestion,XBP1s is an anti-lipogenic protein and that increasing XBP1sactivity reduces hepatic TG content in two different and rele-vant mouse models of obesity and NAFLD. Interestingly, theanti-lipogenic activity of XBP1s is mediated independently ofits transcriptional activity. These results indicate that XBP1screates the anti-lipogenic effect through a protein-proteininteraction. It will require further research to unravel theunderlying molecular mechanism of the anti-lipogenic activityof XBP1s.

Fatty liver results from increased de novo synthesis anduptake of fatty acids, which are subsequently esterified in TG,and reduced TG catabolism because of reduced lipolysis andexport of TG to the circulation (9). XBP1s consistently induced

plasma TG levels in all mouse models described in this manu-script. This is in line with previous reports showing that deple-tion of hepatic XBP1s reduced TG secretion (24, 44). We pro-pose that enhanced breakdown of hepatic lipid droplets andreduction in hepatic TG storage leads to redistribution of lipidsfrom the liver to the periphery, thereby contributing to the risein plasma TG levels in DIO and ob/ob mice.

Macrolipophagy has been suggested previously as an initiat-ing step in lipolysis (34). Indeed, electron microscopy experi-ments performed in this study also indicate that macrolipo-phagy is increased in XBP1s-overexpressing mice and aresuggestive of increased breakdown of lipid droplets. Biochem-ical experiments have strengthened this conclusion. IncreasedATG5 levels in liver homogenates and increased LC3BII levelsin isolated hepatic lipid droplets further confirm thatautophagy of lipid droplets was indeed increased in adXBP1s-injected mice. In response to starvation, LC3BII associationwith lipid droplets and subsequent initiation of macrolipo-phagy is increased in healthy, lean mice, and this process isblunted in DIO mice (34). Excess nutrient supply, such as pro-longed HFD feeding, has been reported to reduce autophagyefficiency, thereby contributing to the development of ER stressand insulin resistance (45– 47). The vicious cycle in whichincreased cellular lipid content impairs macrolipophagy, whichthen further reduces autophagic function, eventually leads tomore lipid retention. It is important to note that XBP1s activityis severely reduced in the liver of obese mice (13), where there ispronounced NAFLD, compared with lean mice. Our resultsindicate that increased XBP1s activity interrupts this defectivemacrolipophagy cycle by reinstating macrolipophagic activityand potentially increasing fatty acid availability for redistribu-tion to the periphery, as shown by increased plasma TG levels inadXBP1s-injected mice. Taken together, the electron micros-copy and biochemical results obtained from analysis of ATG5and LCB3II levels strongly support that XBP1s promotes lipiddroplet breakdown in the liver.

In addition to lowering hepatic TG levels, XBP1s reducedhepatic DAG content in DIO mice. DAGs are second messen-gers that activate members of the novel PKC family of proteins(26). Intracellular compartmentalization of DAGs has beenindicated to play a critical role in activation of PKC� (27–29, 32,33). We report that DAGs are specifically reduced in the cyto-plasmic compartment of livers of XBP1s-expressing mice. Theconcomitant reduction in PKC� activation suggests thatXBP1s-mediated reduction in cytoplasmic DAGs impairsPKC� activation. PKC� has been reported to be enhanced infatty livers of insulin-resistant rodents (28) and humans (30).Reduction of PKC� activity following reactivation of XBP1s is afurther support for the anti-lipogenic actions of this transcrip-tion factor.

In humans, increased levels of hepatic DAG and PKC� pro-tein levels have been associated with development of insulinresistance (30, 31). PKC� directly interacts with and inhibitsinsulin receptor kinase activity, and depletion of hepatic PKC�improves hepatic insulin action in HFD-fed rats (28). Pkrce�/�

mice are resistant to HFD-induced insulin resistance (48),which further provides evidence for a link between PKC�action and development of insulin resistance. Reducing

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PKC� activation, as indicated by reduced cytoplasm-to-membrane translocation, is a novel mechanism for XBP1s toincrease insulin sensitivity and thereby contribute toimproved glucose homeostasis.

In fructose-fed mice, however, hepatic TG content remainedunaffected by XBP1s expression despite significantly reducedlipogenic gene expression and enhanced plasma TG levels. It isinteresting to note that we did not observe any alterations inXbp1s levels during the high-fructose diet feeding, which indi-cates that isolated high-fructose diet feeding and developmentof fatty liver in this setting might not be associated with ERstress and XBP1s. Furthermore, the underlying core mecha-nism(s) for development of NAFLD under isolated high-fruc-tose diet feeding could be overcoming the anti-lipogenic effectsof XBP1s, even if XBP1s is exogenously activated in this setting.Thus, we conclude that lack of ER stress, obesity, and insulinresistance in fructose-fed mice versus DIO and ob/ob miceexplains the lack of effect of XBP1s in this model.

Collectively, our results indicate that XBP1s is an anti-lipo-genic protein and that hepatic reactivation of XBP1s in obesitydoes not create fatty liver but, on the contrary, reduces NAFLDin mice. Approaches that aim to reactivate XBP1s in the liverwill therefore reveal crucial insights for the development oftherapeutic options for XBP1s-mediated regulation of NAFLD.

Experimental Procedures

Reagents—XBP1, PKC�, GAPDH, ADRP, and HRP-conju-gated goat anti-mouse and goat anti-rabbit antibodies werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA).FoxO1, NUP98, FasN, LC3B, NaATPase, and �-tubulin anti-bodies were from Cell Signaling Technology (Beverley, MA).ATG5 antibody was from Novus Biologicals (Littleton, CO).Free glycerol reagent and the serum triglyceride determinationkit were from Sigma-Aldrich (St. Louis, MO). Infinity total cho-lesterol reagent was from Fisher Scientific (Waltham, MA). Thedetergent-compatible protein assay kit, SYBR Green Supermix,and cDNA synthesis kit were from Bio-Rad. BM chemilumines-cence blotting substrate was from Roche. The ultra-sensitivemouse insulin ELISA kit was from Crystal Chem.

Adenovirus Production and Injection—Adenovirus was gen-erated using the ViraPower adenoviral expression system(Invitrogen) as described previously (13). Crude adenovirus wasamplified, CsCl-purified, and titrated by Vector Biolabs(Malvern, PA). Prior to injection, the adenovirus was thawed atroom temperature, and injection solutions were prepared in100 �l of sterile NaCl (8*10E7 pfu/g). Mice were restrained in arestrainer, and adenovirus was injected through the tail veinusing a 28-gauge needle. Mild pressure was applied on the siteof injection to prevent backflow of the injected solution.

Animals and Diets—Wild-type C57BL6/J and leptin-defi-cient ob/ob mice (on a C57BL6/J background) were purchasedfrom The Jackson Laboratory (Bar Harbor, ME). Mice werehoused in a temperature-controlled, air-conditioned, and spe-cific pathogen-free animal facility at Boston Children’s Hospi-tal. Mice were housed in groups (5 mice/cage) and maintainedon a 12:12-h light/dark cycle (lights on at 7 a.m.). Diet-inducedobesity was established by feeding wild-type mice a high-fat diet(HFD) (45% kcal, Research Diets Inc., New Brunswick, NJ) for

12 weeks prior to starting the experiments. A fructose diet (60%kcal, TD 00202, Teklad, Harlan Laboratories, Madison, WI)was provided for 4 weeks prior to starting the experiments. TheHFD and especially the fructose diet are hygroscopic and weretherefore fully replaced every week. Ob/ob mice were fed stan-dard laboratory chow. Mice had ad libitum access to food andwater. All animal experiments were approved by the AnimalCare and Use Committee of Boston Children’s Hospital.

Glucose and Insulin Tolerance Test—For glucose tolerancetesting, mice were fasted overnight (7p.m. to 8 a.m.). Bloodglucose was measured from the tail vein prior to an i.p. injectionof 1 g/kg D-glucose in 200 �l of sterile NaCl. Glucose levels weremeasured from the tip of the tail using a glucometer (Bayer) atindicated time points. For insulin tolerance testing, food wasremoved for 6 h (8 a.m. tp 2 p.m.). Blood glucose was measuredfrom the tip of the tail prior to an i.p. injection of 0.75 IU/kginsulin (recombinant human insulin, Eli Lilly) in 200 �l of ster-ile NaCl. Glucose levels were measured from the tip of the tail atindicated time points.

Blood and Tissue Collection—Mice were terminated by car-diac puncture under isoflurane anesthesia after a 6-h fast (8 a.m.to 2 p.m.). Blood was collected in heparin-coated tubes and kepton ice until centrifugation (8000 � g, 4 °C, 20 min). Plasma wasaliquoted and used for analysis immediately or stored at�80 °C. Tissues were quickly excised and snap-frozen in liquidnitrogen or fixed for microscopic analysis.

Electron Microscopy—Fresh liver was cut and immediatelyfixed in 2.5% glutaraldehyde-buffered solution at room temper-ature for 2 h and then at 4 °C overnight. After washing with PBSthree times, tissues were post-fixed in 2% OsO4, dehydrated,and infiltrated. Samples were sectioned and photographed atthe Electron Microscopy/Plastic Core at the Joslin DiabetesCenter (Boston, MA).

H&E Staining—Fresh liver was cut and immediately fixed in10% buffered formalin at 4 °C overnight. Tissues were paraffin-embedded, cut, and mounted onto glass slides prior to H&Estaining according to the standard protocol of the RodentHistopathology Core Facility of Harvard Medical School(Boston, MA).

Tissue Lysis and Western Blotting—Liver tissue (75 mg) washomogenized in ice-cold lysis buffer (25 mM Tris-HCl (pH 7.4),100 mM NaF, 50 mM Na4P2O7, 10 mM Na3VO4, 10 mM EGTA,10 mM EDTA, 1% Nonidet P-40, 2 mM PMSF, and proteaseinhibitor mixture) using a TissueLyserII (Qiagen). Homoge-nates were incubated on ice for 20 min and centrifuged at16,100 � g at 4 °C for 1 h. The lipid layer was carefully removed,and supernatants were centrifuged for another hour (16,100 �g, 4 °C). Samples were denatured in 1� Laemmli buffer by boil-ing at 100 °C for 5 min. Nuclear proteins were extracted from100 mg of liver tissue using a commercially available kit (NE-PER nuclear and cytoplasmic extraction kit, Fisher Scientific)according to the protocol of the manufacturer. Equivalentamounts of proteins from each sample were resolved by SDS-PAGE and then electrotransferred onto PVDF membranes.The membranes were blocked in 10% blocking reagent pro-vided with BM chemiluminescence blotting substrate (peroxi-dase) in TBS (pH 7.4) for 1 h at room temperature. The mem-branes were subsequently incubated with primary antibody in

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5% blocking reagent (in TBS Tween (pH 7.4)) overnight at 4 °C.Membranes were washed three times for 20 min in TBST andincubated with secondary antibody in 5% blocking reagent (inTBS Tween (pH 7.4)) for 1 h at room temperature. After exten-sive washing, the blots were developed using a chemilumines-cence assay system (Roche) and exposed to Kodak films. Themembranes were stripped in stripping buffer (62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptomethanol) at50 °C for 20 min under vigorous shaking. Densitometry usingImageJ software (National Institutes of Health) was conductedfor protein quantification on the immunoblots.

Plasma and Liver Lipid Analysis—Plasma triglycerides andcholesterol were measured using commercially available color-imetric reagents (Wako, Richmond, VA). Neutral lipids wereisolated from liver homogenates (75 mg of tissue in 1 ml of 50mM NaCl) using chloroform/methanol (2:1 v/v).The interphasewas washed with 50 mM NaCl once, and aliquots of the organicphase were supplemented with 10% Triton X-100 (Sigma) anddried. Triglyceride and cholesterol content was measured onthe dried pellet directly using commercially available colori-metric reagents (Wako). DAG and ceramide were isolated from100 mg of liver and prepared for LC/MS/MS analysis asdescribed previously (49). Total DAG and ceramides were cal-culated from the sum of individual species.

In Vivo Fatty Acid Synthesis Assay—The fatty acid synthesisrate was determined using a protocol described previously withminor modifications (50, 51). Randomly fed mice were weighedand intraperitoneally injected with 2 mCi [3H2O] as a chaser.1 h after chaser injection, the liver was harvested, briefly dried,weighed, and snap-frozen in liquid nitrogen. Liver tissues wereincubated in 2.5 M KOH at 75 °C for 2 h. After cooling down toroom temperature, 2 volumes of 80% ethanol and 4 volumes ofhexane were added to the samples and mixed completely. Themixture was separated by centrifugation, and the aqueousphase was saved. After acidification with 3 M H2SO4, fatty acidswere extracted from the aqueous phase using hexane andwashed with 50 mM H2SO4 twice. Fatty acids were nitrogen-dried and solubilized in 500 �l of chloroform. Samples wereloaded onto TLC plates with hexane:diethyl ether:glacial aceticacid (70:20:10 v/v/v) as liquid phase. Fatty acid spots on theTLC plates were visualized in iodine vapor and cut out for liquidscintillation measurements. The rate of hepatic fatty acids wascalculated as the amount of [3H] incorporated into fatty acidsper gram of tissue per hour.

Protein Isolation from Liver Lipid Droplets—One entireob/ob mouse liver was used to extract lipid droplet proteins foreach sample using an adapted protocol (34). Livers werehomogenized in 0.25 M sucrose supplemented with proteaseinhibitors on ice. Liver lysates were centrifuged at 6800 � g at4 °C for 10 min, and supernatants were centrifuged again at17,000 � g at 4 °C for 10 min. Supernatants and fatty layer werecollected and adjusted to 20% sucrose in a hypotonic lysis buffer(20 mM Tris-HCl (pH 7.4) and 1 mM EDTA) and subjected togradient centrifugation at 28,000 � g at 4 °C for 30 min. Fattylayers containing lipid droplets were collected and delipidatedwith organic solvents. Briefly, lipid droplets were mixed with 10volumes of acetone prechilled at �80 °C and incubated at�20 °C overnight. Following centrifugation at 4300 � g at 4 °C

for 1 h, pellets were washed with 10 volumes of acetone, 10volumes of 1:1 acetone/ether, and 10 volumes of ether sequen-tially. Dried pellets were thoroughly resuspended in samplebuffer (130 mM Tris-HCl (pH 6.8), 4% SDS, and 4% 2-mercap-toethanol) and incubated at 60 °C for 5 h in a sonicating waterbath. Dissolved samples were centrifuged at 18,500 � g at roomtemperature for 10 min, and supernatants were analyzed byWestern blotting.

Reverse Transcription and Quantitative Real-time PCR—Total RNA was extracted using QIAzol reagent (Qiagen, Valen-cia, CA). 1 �g of RNA was reverse-transcribed to cDNA using acDNA synthesis kit (Bio-Rad) under the following conditions:25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min. Tran-scripts were diluted 20 times, and QPCR analysis was per-formed using 4 �l of cDNA. Gene expression was analyzedusing SYBR Green Supermix (Bio-Rad) on an iQ5 multicolorreal-time PCR detection system (Bio-Rad) using the followingprimers: 18s rRNA forward, 5-AGT CCC TGC CCT TTGTAC ACA-3; 18s rRNA reverse, 5-CGA TCC GAG GGC CTCACT A-3; Xbp1s forward, 5-GGT CTG CTG AGT CCG CAGCAG G-3; Xbp1s reverse, 5-AGG CTT GGT GTA TAC ATGG-3; Pkrce forward, 5-GAG GAC TGG ATT GAC CTGGA-3; Pkrce reverse, 5-ATC TCT GCA GTG GGA GCAGT-3; Srebp1c forward, 5-GCG GTT GGC ACA GAG CTT-3; Srebp1c reverse, 5-GGA CTT GCT CCT GCC ATC AG-3;Acc1 forward, 5-ATT GGG CAC CCC AGA GCT A-3; Acc1reverse: 5-CCC GCT CCT TCA ACT TGC T-3; Acc2 for-ward, 5-GGG CTC CCT GGA TGA CAA C-3; Acc2 reverse,5-TTC CGG GAG GAG TTC TGG A-3; FasN forward,5-GGA GGT GGT GAT AGC CGG TAT-3; FasN reverse,5-TGG GTA ATC CAT AGA GCC CAG-3; Scd1 forward,5-AGA TCT CCA GTT CTT ACA CGA CCA C-3; Scd1reverse, 5-GAC GGA TGT CTT CTT CCA GGT G-3; Dgat2forward, 5-TTC CTG GCA TAA GGC CCT ATT-3; Dgat2reverse, 5-AGT CTA TGG TGT CTC GGT TGA C-3; Erdj4forward, 5-GCA ATG GGA GTC CTT TTG AA-3; Erdj4reverse, 5-CCT GGA AGT GAT GCC TTT GT-3; Grp58 for-ward, 5-TAT GAT GGG CCTA GGA CTG C-3; Grp58reverse, 5-TGC TGG CTG CTT TTA GGA AT-3; Grp78 for-ward, 5-AGT GGT GGC CAC TAA TGG AG-3; and Grp78reverse, 5-CAA TCC TTG CTT GAT GCT GA-3.

Statistical Analysis—Data are presented as mean � S.E. Sta-tistical significance was calculated using two-tailed Student’s ttest or two-way analysis of variance testing with Bonferronipost-test for multiple-comparison analysis (for GTT and ITT)and was accepted at *, p � 0.05; **, p � 0.01; and ***, p � 0.001.

Author Contributions—Conceptualization, H. H. and U. O.; Meth-odology, H. H., Y. Z., G. I. S., and U. O.; Investigation, H. H., Y. Z.,D. Z., J. L., and M. A. S. H.; Writing—Original Draft, H. H. and U. O.;Writing—Review & Editing, H. H., G. I. S., and U. O.; Supervision,H. H. and U.O.; Funding Acquisition, U. O.

Acknowledgments—We thank Drs. Jonathon N. Winnay and C. Ron-ald Kahn (Joslin Diabetes Center, Harvard Medical School) for assis-tance with the in vivo fatty acid synthesis assay.

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XBP1s Is an Anti-lipogenic Protein

17404 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

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Hernandez, Gerald I. Shulman and Umut OzcanHilde Herrema, Yingjiang Zhou, Dongyan Zhang, Justin Lee, Mario Andres Salazar

XBP1s Is an Anti-lipogenic Protein

doi: 10.1074/jbc.M116.728949 originally published online June 20, 20162016, 291:17394-17404.J. Biol. Chem. 

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