Reduction of Hypothalamic Endoplasmic Reticulum Stress Activates Browning of White Fat and Ameliorates Obesity

Energy balance can be regulated by peripheral signals acting on the central nervous system (CNS), including the hypothalamus (1–3). The number of studies on this topic is continuously increasing, with the aim to identify new therapeutic approaches against obesity. Increased energy expenditure is one way to reduce body weight (4–6), and in recent years, there has been a growing interest in the activation of the thermogenic process, especially in the central control of brown adipose tissue (BAT) activity (3,7–10). Even more recently, activation of beige/brown-in-white (brite) adipocytes in the white adipose tissue (WAT), a process known as browning, has been recognized as a therapeutic target to increase energy expenditure and reduce body weight (6,11–13).

The endoplasmic reticulum (ER) is a cellular compartment where proteins are matured, assembled, and folded, and any alteration in ER homeostasis that disturbs this protein processing will lead to accumulation of unfolded proteins, which in turn triggers the ER unfolding protein response (UPR) (14–17). Increasing evidence has shown a strong interaction between ER stress and the pathology of obesity. ER stress is closely related with obesity-associated insulin resistance in peripheral tissues, such as the pancreas and liver (18–20). Hypothalamic ER stress occurs under conditions of nutritional excess, such as obesity and type 2 diabetes, leading to leptin and insulin resistance (21–25). There is a potential therapeutic interest in this mechanism because recent data demonstrated that amelioration of hypothalamic ER stress, through genetic or pharmacologic improvement of protein folding, reduces body weight and improves leptin and insulin sensitivity (21–23,26,27). In addition to the effect of ER stress on leptin and insulin resistance, recent data have also shown that central ceramide–induced lipotoxicity induces ER stress leading to weight gain, glucose intolerance, decreased sympathetic tone, and BAT thermogenesis (23,28). Notably, the central action of ceramides can be reversed by decreasing ER stress, specifically in the ventromedial nucleus of the hypothalamus (VMH) (23), a key site modulating BAT thermogenesis (3,9,10).

Despite the evidence linking hypothalamic ER stress to several metabolic actions, its potential effect on browning of WAT and its eventual (patho)physiologic relevance remains unknown. Similarly, the central role of GRP78 in the process and in the control of BAT thermogenesis remains unknown. Therefore, in this study we aimed to determine the importance of hypothalamic ER stress and GRP78 function on browning of WAT and the BAT thermogenic program.

Male Sprague-Dawley rats (100 g, 4–5 weeks of age; Animalario General Universidade de Santiago de Compostela, Santiago de Compostela, Spain) and obese Zucker rats (OZR) (fa/fa; 300–350 g) and their controls, lean Zucker rats (LZR) (fa/–; 250–300 g), both provided by Charles River Laboratories (Barcelona, Spain), were used for the experiments. Animals were housed on a 12-h light (0800 h–2000 h), 12-h dark cycle, in a temperature- and humidity-controlled room and maintained with chow and water ad libitum. Sprague-Dawley rats were distributed to two main feeding groups: 1) a standard laboratory diet (STD) (SAFE A04: 3.1% fat, 59.9% carbohydrates, 16.1% proteins, 2.791 kcal/g; Scientific Animal Food & Engineering, Nantes, France), and 2) a high-fat diet (HFD) (D12451: 45% fat, 35% carbohydrate, 20% protein, 4.73 kcal/g; Research Diets, Inc., New Brunswick, NJ). The animals were housed collectively (n = 4 animals/cage) during 3–6 months with these diets until the experimental procedures started. After HFD, during all the experimental procedures, animals were individually housed, and their respective food intake (STD or HFD) and body weight were monitored daily by the researchers. The experiments were performed in agreement with the International Law on Animal Experimentation and were approved by the Universidade de Santiago de Compostela Ethical Committee (Project License 15010/14/006).

Chronic intracerebroventricular (ICV) cannulae were implanted under ketamine-xylazine anesthesia in STD or 3-month HFD rats, as described previously (1,7,8,23,29–32). Animals were individually caged and allowed to recover for 4 days, and then the rats were ICV administered the chemical chaperone tauroursodeoxycholic acid (TUDCA) (10 µg/day; Calbiochem, Billerica, MA) (21,22,27) or vehicle (PBS), or were administered the ER stress-inductor thapsigargin (0.2 µg/day; Sigma-Aldrich, St. Louis, MO) or vehicle (DMSO). Animals were treated during 7 days, with body weight and food intake daily recorded. For the leptin resistance test, leptin (10 µg; Sigma-Aldrich) was ICV administered in rats fasted overnight (12 h).

Rats were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) under ketamine-xylazine anesthesia (50 mg/kg, i.p.). The VMH was targeted bilaterally using a 25-gauge needle (Hamilton, Reno, NV). The injections were directed to the following stereotaxic coordinates for the VMH: 2.4/3.2 mm posterior to the bregma, ±0.6 mm lateral to midline and 10.1 mm ventral, as previously reported (7,8,23,30,32). Adenoviral (Ad) vectors (Viraquest, North Liberty, IA) containing GFP (used as control) or GRP78 and GFP (10¹² parts/mL) were delivered at a rate of 200 nL/min for 5 min (1 µL/injection site), as previously reported (7,8,23,30,32). After the Ad treatment, animals were monitored for 7 days. We used 8–10 rats per group, and the experiment was repeated six times because of the diverse experimental approaches. The β3 adrenergic receptor (β3-AR)–specific antagonist SR59230A ([3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol-oxalate]; 3 mg/kg/day dissolved in saline/DMSO [1:50]; Tocris Bioscience, Bristol, U.K.) (7,8) was administered subcutaneously, and animals were treated for 2 days before the GRP78 injections.

Body temperature was recorded twice at the end of the treatments with a rectal probe connected to a digital thermometer (BAT-12 Microprobe Thermometer; Physitemp Instruments, Inc., Clifton, NJ). Skin temperature surrounding BAT was recorded with a B335 compact infrared thermal imaging camera (FLIR Systems, West Malling, Kent, U.K.) and analyzed with FLIR Tools software (FLIR Systems) (8,23,30–32).

Rats were killed by cervical dislocation and decapitation. From each animal the VMH, liver, the interscapular BAT (iBAT), the gonadal WAT (gWAT), and the subcutaneous WAT (sWAT) (from the inguinal area) were collected for Western blotting, Oil Red O staining, and real-time PCR analysis and immediately homogenized on ice to preserve phosphorylated protein levels. Those samples and the serum were stored at −80°C until further processing. Dissection of the VMH was performed by micropunch procedure under the microscope, as previously described (7,8,23,30). Samples from gWAT and sWAT were immersed in 10% formalin for 24 h and then in ethanol 70% for immunohistochemical analysis.

Serum nonesterified fatty acids (NEFAs) concentrations were determined using a Wako NEFA-HR Kit (Wako Chemicals GmbH, Neuss, Germany), and triacylglyceride and cholesterol levels were determined using a Spinreact Kit (Spinreact S.A., San Esteve de Bas, Spain). Serum insulin levels were measured using a Rat/Mouse Insulin ELISA kit EZMRI-13K (Millipore, Billerica, MA). Leptin levels were determined using a Rat Leptin ELISA kit EZRL-83K (Millipore). All of the methods used have been reported previously (23,33).

Dissected VMH, BAT, and WAT were homogenized and lysed with buffer with the following composition: Tris-HCl (pH 7.5) 50 mmol/L, EGTA 1 mmol/L, EDTA 1 mmol/L, Triton X-100 1% vol/vol, sodium orthovanadate 0.1 mmol/L, sodium fluoride 50 mmol/L, sodium pyrophosphate 5 mmol/L, sucrose 0.27 mol/L, and protease inhibitor cocktail, as previously shown (1,7,8,23,30–32). Protein lysates were subjected to SDS-PAGE, electrotransferred on a polyvinylidene fluoride membrane, and probed with the following antibodies: GRP78 (1:1,000; ref. 3183), phosphorylated (p) protein kinase B (pAKT) (Ser473; 1:1,000; ref. 9271), and phosphorylated phosphatidylinositide 3-kinase (pPI3K_p85) (Tyr458; 1:1,000; ref. 4228) from Cell Signaling (Danvers, MA); activating transcription factor 6-α (ATF6α) (1:1,000; ref. sc-22799), phosphorylated PKR-like ER kinase (pPERK) (Thr981; 1:500; ref. sc-32577), phosphorylated eukaryotic initiation factor 2-α (peIF2α) (Ser52; 1:2,000; ref. sc-101670), and C/EBP homologous protein (CHOP) (1:1,000; ref. sc-793) from Santa Cruz Biotechnology (Santa Cruz, CA); phosphorylated inositol-requiring enzyme 1-α (pIRE1α) (Ser724; 1:1,000; ref. ab48187), phosphorylated signal transducer and activator of transcription 3 (pSTAT3) (Tyr705; 1:1,000; ref. ab76315), and uncoupling protein-1 (UCP1) (1:10,000; ref. ab10983) from Abcam (Cambridge, U.K.); and β-actin (1:5,000; ref. A5316) and α-tubulin (1:5,000; ref. T5168) from Sigma-Aldrich, as previously described (7,8,23,30,31,33). Values were expressed in relation to α-tubulin (for BAT and WAT) or β-actin (for hypothalamus) protein levels.

We performed real-time PCR (TaqMan; Applied Biosystems, Carlsbad, CA), as previously described (1,7,8,23,30,31,33), using specific sets of primers and probes (Supplementary Table 1). Values were expressed relative to hypoxanthine-guanine phosphoribosyltransferase (HPRT) levels.

Detection of UCP1 in WAT was performed using anti-UCP1 (1:500; ab10983; Abcam), as previously reported (34). Hepatic lipid content was analyzed by Oil Red O staining, as previously shown (23,33). Images were taken with an Olympus XC50 digital camera (Olympus Corp., Tokyo, Japan) at original magnification ×20. Digital images for liver and WAT were quantified with ImageJ software (National Institutes of Health, Bethesda, MD). Direct detection of GFP fluorescence was performed after perfusion of the animal and detected with an Olympus IX51 fluorescence microscope at original magnification of ×4.

Glycemia was measured at 0, 15, 30, and 60 min after insulin or 0, 30, 60, 90, and 120 min after glucose administration with an Accu-Check glucometer (Roche, Barcelona, Spain), after an intraperitoneal injection of 0.75 units/kg insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) for insulin tolerance test (ITT) or 2 mg/g d-glucose (Sigma-Aldrich) administered orally via gavage for glucose tolerance test (GTT) (23). Animals were fasted overnight for the GTT. HOMA insulin resistance (IR) was calculated as follows: HOMA-IR = (FPI × FPG)/22.5, with FPI indicating fasting plasma insulin (mU/L) and FPG indicating fasting plasma glucose (mmol/L).

Data are expressed as mean ± SEM. mRNA and protein data were expressed in relation (%) to control (STD, GFP, or vehicle) rats. Statistical significance was determined by Student t test. P < 0.05 was considered significant.

Rats fed the HFD showed increased body weight and energy intake (Fig. 1A and B). ER UPR proteins, such as GRP78/BiP (glucose-regulated protein 78 kDa/binding immunoglobulin protein), pIRE1, pPERK, peIF2-α, ATF6-α, and CHOP, were increased in the VMH of the HFD group, demonstrating an augmentation of ER stress (Fig. 1C).

We aimed to investigate whether amelioration of hypothalamic ER stress might revert or improve the metabolic phenotype of diet-induced obese (DIO) rats. Therefore, rats fed the HFD were treated ICV with the chemical chaperone TUDCA (21,22,27). Central administration of this drug to HFD (but not STD) rats induced feeding-independent weight loss (Fig. 2A and B), decreased hypothalamic ER stress (Supplementary Fig. 1A and B) and increased body temperature (Fig. 2C), BAT temperature (Fig. 2D), BAT darkness (Fig. 2E), and UPC1 protein levels in BAT (Fig. 2F), gWAT (Fig. 2G), and sWAT (Fig. 2H), the latter being indicative of browning (6,11–13,35). Of note, none of these metabolic changes were found in the rats fed the STD (Fig. 2A and C–H).

The chaperone GRP78/BiP, located in the ER, facilitates the protein folding upstream of the ER UPR (14,36). Thus, an Ad encoding GRP78 or control Ads expressing GFP alone were injected into the VMH, a key site modulating BAT (3,9,10). Infection efficiency in the VMH was assessed by the expression of GFP (Fig. 3A) and also by increased concentration of GRP78 in the VMH (Fig. 3B and C). GRP78 Ads elicited a decrease in the protein levels of pIRE, peIF2-α, and CHOP in the VMH of STD rats (Fig. 3B) and reduced peIF2-α and CHOP in 3-month HFD rats (Fig. 3C). Moreover, administration of GRP78 Ads into the VMH induced feeding-independent weight loss in HFD rats but not in STD rats (Fig. 3D and E). To better control the specificity of VMH administration, we performed “missed” GRP78 injections, in “VMH neighboring” areas (Supplementary Fig. 2A). Our anatomical data showed that the VMH was not hit, as indicated by the absence of GFP in this nucleus. Notably, no changes in body weight (Supplementary Fig. 2B) or food intake (Supplementary Fig. 2C) were found under these conditions. This evidence indicates that the effect of GRP78 on body weight was specific to the VMH and not related to spreading.

In keeping with these data, injection of GRP78 Ads led to reduced size of the subcutaneous fat pad in HFD rats but not in STD rats (Fig. 3F and G). These effects were associated with an improvement in the metabolic phenotype of the HFD rats, as demonstrated by decreased hepatic steatosis (Fig. 3H). Also, in relation to this, cholesterol serum levels were reduced and leptin serum levels showed a trend, albeit a statistically nonsignificant one, to be decreased by the effect of GRP78 Ads on HFD rats but with no changes in triacylglyceride, circulating NEFAs, or insulin (Supplementary Table 2).

To investigate whether the magnitude of DIO could affect the effect of hypothalamic GRP78 manipulation, we generated a 6-month HFD model. Treatment with Ads harboring GRP78 within the VMH recapitulated the effects observed in the 3-month HFD model: a feeding-independent (massive) decrease in body weight (Supplementary Fig. 3A and B), associated with reduced adiposity in sWAT and a nonsignificant trend in gWAT (Supplementary Fig. 3C).

We evaluated the effect of VMH GRP78 overexpression on peripheral glucose homeostasis and insulin sensitivity in STD and 3-month HFD rats. Our data showed that administration of GRP78 into the VMH did not affect glucose tolerance in STD (Fig. 4A) or HFD rats (Fig. 4B). However, the insulin resistance that characterizes HFD rats was improved by GRP78 Ad in the VMH (Fig. 4D and F) but did not affect STD rats (Fig. 4C and E). Calculation of HOMA-IR confirmed that HFD-induced insulin resistance was ameliorated by VMH GRP78 (STD Ad GFP: 1.42 ± 0.02; STD Ad GRP78: 1.48 ± 0.03; HFD Ad GFP: 2.90 ± 0.05 [P < 0.001] vs. STD Ad GFP; HFD Ad GRP78: 2.11 ± 0.02 [P < 0.001] vs. HFD Ad GFP). Similar data were obtained when GRP78 was administered to 6-month HFD rats (Supplementary Fig. 3D–F).

ER stress is known to induce leptin and insulin resistance (21–23). Our data showed that in the VMH of 3-month HFD, but not STD rats, pSTAT3, pAKT, and pPI3K protein levels were increased after the administration of GRP78 (experiment in basal conditions, no previous leptin/insulin stimulation) (Fig. 4G and H). Similar data were obtained in the 6-month HFD model (data not shown). To add functional insight to the data, ICV leptin was given to HFD rats treated with Ad GFP or Ad GRP78 in the VMH, and food intake was measured at 12 h. Although ICV leptin had a potent anorectic action in STD rats, this effect was gone in HFD rats. Importantly, and in keeping with the molecular data, injection of Ad GRP78 in the VMH recovered leptin sensitivity in HFD rats (Fig. 4I).

Next, we investigated the effect of GRP78 Ads on thermogenesis. Our data showed that although GRP78 in the VMH did not affect the temperature of the BAT or the body temperature in STD rats (Fig. 5A and D), it did increase BAT temperature and trended to increase body temperature in 3-month HFD rats (Fig. 5B and F). In line with this, GRP78 Ads increased the BAT darkness (Fig. 5C and E) and the UCP1 protein levels (Fig. 5G and H) in the BAT of HFD but not STD rats. Notably, similar effects were observed when GRP78 was administered to 6-month HFD rats (Supplementary Fig. 4A–D).

After demonstrating that administration of GRP78 Ads into the VMH activates BAT thermogenesis in HFD rats, we explored the possibility that it may also regulate browning of WAT, as we observed with central TUDCA (Fig. 2G and H). The size of the adipocytes was larger in both gWAT and sWAT from 3-month HFD rats compared with STD rats, and the treatment with GRP78 into the VMH reduced significantly adipocyte size in both diet regimens (Fig. 5I and J). The administration of GRP78 in the VMH of HFD rats increased UCP1 immunostaining in the gWAT compared with GFP HFD rats (Fig. 5I), which was also observed in the sWAT, although the tendency was not significant (P = 0.1) (Fig. 5J). Comparable data were found when the same samples were analyzed by Western blot, which confirmed the elevation in UCP1 expression in the sWAT and gWAT of HFD rats, but not STD rats, treated with GRP78 Ads in the VMH (Fig. 5K and L). The same overall effects on the size of the gonadal and inguinal adipocytes and UCP1 immunostaining (Supplementary Fig. 4E and F) were displayed by 6-month HFD rats treated with Ads encoding GRP78, which also showed increased expression of other browning markers, such as peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) in HFD rats, and PR domain-containing 16 (PRDM16) in both STD and HFD rats, but not cell death-inducing DFFA-like effector a (CIDEA) in the white fat (Supplementary Fig. 4G and H).

BAT thermogenesis is mainly controlled by the sympathetic nervous system (SNS) via β3-AR (3,37,38). Thus, we investigated whether regulation of BAT after administration of GRP78 Ad particles in the VMH was mediated by the SNS. Pharmacologic inactivation of β3-AR by subcutaneous administration of the specific antagonist SR59230A (7,8) prevented the effect on body weight associated with central administration of GRP78 viruses (Fig. 6A) without interfering with feeding (Fig. 6B). Consistent with the increased weight gain after the β3-AR blockade, the amount of inguinal and gonadal fat was augmented (Fig. 6C). The treatment with SR59230A induced a decrease in BAT darkness and promoted accumulation of WAT in the interscapular fat pad (Fig. 6D). Furthermore, SR59230A decreased UCP1 protein levels (Fig. 6E), as well as body and BAT temperature (Fig. 6F and G).

Recent evidence has shown that browning is modulated by SNS via β3-AR in the white adipocytes (6,9). Thus, we investigated the effect of SR59230A on WAT browning. SR59230A reversed the brown-like phenotype of WAT induced by GRP78 Ad particles, as demonstrated by increased adipocyte area and decreased UCP1 immunostaining (Fig. 6H and J) and UCP1 protein levels (Fig. 6I and K) in both gWAT and sWAT.

We also aimed to investigate whether the central effect of VMH GRP78 on browning was direct or secondary to leptin action because of GRP78-induced weight loss, decreased adiposity, and a tendency to diminish leptin levels (Fig. 3F and G, Supplementary Fig. 3C, and Supplementary Table 2). To address this, we analyzed the effect of GRP78 Ad particles in OZRs and their LZR littermates. Our data showed that GRP78 treatment into the VMH promoted feeding-independent weight loss in OZRs but not in their LZR littermates (Supplementary Fig. 5A and B). Furthermore, GRP78 Ads into the VMH promoted browning, as demonstrated by decreased adipocyte area and increased UCP1 immunostaining (Fig. 7A and B), as well as UCP1 protein levels (Fig. 7C and D), in the gWAT or the sWAT of OZR but not in LZR.

Finally, we wanted to investigate whether the effects of GRP78 manipulation in the VMH could be detectable in another model of ER stress; namely, that induced pharmacologically by central administration of thapsigargin. Central treatment with this drug induced feeding-independent weight gain (Fig. 8A and B), increased hypothalamic ER stress (Fig. 8C), decreased BAT darkness, and increased fat accumulation (Fig. 8D), a trend to decrease body temperature (Fig. 8E), and a significant decrease in BAT temperature (Fig. 8F) as well as decreased UPC1 protein levels in BAT (Fig. 8G), gWAT (Fig. 8H), and sWAT (Fig. 8I). Of note, all of those responses were reversed or improved by administration of GRP78 Ad into the VMH (Fig. 8A–I). Overall, this evidence demonstrates that by attenuating ER stress, GRP78 in the VMH is an important modulator of energy balance by controlling both BAT thermogenesis and browning of WAT.

Here, we show that the chaperone GRP78/BiP acts within the VMH, a key nucleus-modulating energy expenditure (3,9,10), to exert a beneficial effect on HFD-induced obesity and thapsigargin-induced weight gain via the activation of BAT thermogenesis and the induction of browning of white fat. This central mechanism is mediated through the SNS and acts also in OZRs, suggesting at least some independence of leptin.

Overnutrition and lipotoxicity induce ER stress and alter the normal protein folding in peripheral tissues (18–20). Obesity, overnutrition, or elevated ceramide levels promote hypothalamic ER stress and contribute to peripheral leptin and insulin resistance (21–23). Of note, treatment with chemical chaperones that improve protein folding recovers leptin and insulin sensitivity, normalizes feeding, and reduces body weight (21,22,26,27). We found that HFD rats show increased hypothalamic ER stress, as determined by activation of the ER UPR pathway. Central treatment with TUDCA or specific administration of Ad particles encoding GRP78 within the VMH promoted an overall improvement of HFD rats, as shown by decreased adiposity, reduced serum and hepatic lipids, and improved insulin sensitivity. Of note, those effects are associated with increased thermogenesis in BAT and also with browning of white fat.

WAT browning is responsible for a significant increase in total energy expenditure (39), and its stimulation has the therapeutic potential to promote body fat reduction (6,40). Brown adipocytes induced in WAT, also known as “beige” or “brite” cells (6,11,41), are derived from a precursor population distinct from both mature white and brown adipocytes (42,43). Several mechanisms have been proposed for WAT browning (5,6), including prolonged cold exposure (44), adrenergic activation (45–47), and the prostaglandin synthesis enzyme cyclooxygenase 2 (48). However, although multiple factors that modulate the development and function of beige/brite cells have been identified and browning is a sympathetic-driven (and thus hypothalamic-modulated) process (6,9), the role of the CNS in the control of WAT browning remains almost unknown. New evidence has demonstrated that activation of the orexigenic agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus suppresses the browning process and that this effect is mediated by O-linked β-N-acetylglucosamine (12). In addition, leptin and insulin act via proopiomelanocortin neurons to promote browning of white fat (49). Finally, GLP1 agonism in the VMH mediates browning through a mechanism involving inhibition of hypothalamic AMPK (32). Although we have recently shown that ER stress in the VMH decreases SNS activity to inhibit BAT function (23), no data have yet linked hypothalamic ER stress with browning.

In this study, we demonstrate that amelioration of ER stress in the VMH, by genetic targeting of GRP78, leads to increased WAT browning through the activation of sympathetic β3-AR signaling, an action that is associated with weight loss independent of feeding. This action was corroborated in several models: DIO rats (fed the HFD for 3 or 6 months) and rats centrally treated with thapsigargin and OZRs. Of note, this effect seems also independent of leptin signaling, because the reduction of ER stress in the VMH causes weight loss and WAT browning in leptin receptor-deficient OZRs. These results reveal that similar to BAT activation, white fat browning is regulated by ER stress within the VMH and fit with the idea that inhibition of this process (23) protects from DIO (50). Our results are relevant because, besides decreased body weight, treatment with GRP78 Ads elicited a marked overall improvement of the metabolic phenotype of HFD obese rats; that is, decreased adiposity, reduced hepatic steatosis, improved leptin signaling, increased insulin sensitivity, and increased BAT thermogenesis. The specific contribution of beige/brite thermogenesis to the improved metabolic phenotype remains to be established. However, recent evidence has demonstrated that UCP1 in the mitochondria of brite/beige adipose tissue is functionally thermogenic and accounts for about one-third of the interscapular BAT potential (39).

Browning of white fat has therapeutic potential to promote body fat reduction (40). Although several mechanisms have been proposed (12,32,49), the neuronal pathways within the CNS controlling WAT browning remain largely unknown. This study provides evidence that amelioration of ER stress in the VMH by GRP78 is a central mechanism regulating WAT browning. Overall, these data suggest that targeting the hypothalamic control of WAT browning may be a potential strategy against obesity and associated morbidities.

Acknowledgments. The authors thank Tania López-González (Department of Physiology, University of Santiago de Compostela) for her help with the animal work.

Funding. C.C. is a recipient of a Sara Borrel Contract (CD14/0007), I.G.-G. is a recipient of a fellowship from Ministerio de Economía y Competitividad (MINECO) (FPU12/01827), L.L.-P. is a recipient of a fellowship from Xunta de Galicia (ED481A-2016/094), and E.R.-P. is a recipient of a fellowship from MINECO (FPI/BES-2015-072743). The research leading to these results has received funding from the Junta de Andalucía (M.T.-S.: P12-FQM-01943), MINECO cofunded by the FEDER Program of EU (M.T.-S.: BFU2014-57581-P, N.C.: SAF2014-52223-C2-2-R, C.D.: BFU2011-29102, R.N.: BFU2012-35255, M.L.: SAF2015-71026-R and BFU2015-70454-REDT/Adipoplast), Xunta de Galicia (R.N.: EM 2012/039 and 2012-CP069, M.L.: 2015-CP079), the European Community's Seventh Framework Programme (FP7/2007-2013) under ObERStress European Research Council Project grant agreement No. 281854 (M.L.), Instituto de Salud Carlos III (ISCIII) and Fondo Europeo de Desarrollo Regional (FEDER; M.T.-S.: PIE14/00005, M.L.: PI12/01814 and PIE13/00024). CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIII.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.C., I.G.-G., P.S.-C., N.M.-S., L.L.-P., and E.R.-P. performed the in vivo experiments (glucose and insulin tolerance tests and stereotaxic microinjection of adenoviral expression vectors), the analytical methods (immunohistochemistry, serum analyses, Western blotting, real-time PCR), and the analysis of temperature and collected and analyzed the data. C.C., I.G.-G., J.F., M.T.-S., N.C., C.D., R.N., and M.L. analyzed, discussed, and interpreted the data. C.C. and M.L. made the figures. M.L. developed the hypothesis, designed the experiments, coordinated and directed the project, secured funding, and wrote the manuscript. All authors reviewed and edited the manuscript and had final approval of the submitted manuscript. M.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.