Obesity is a chronic metabolic disorder. Some people’s genetics make them more vulnerable to the condition, and it is generally caused by eating too much and moving too little. The resulting surplus of nutrients affects the cells and organs of the body in several adverse ways. For example, excessive nutrients impair a compartment within cells called the endoplasmic reticulum. This compartment is where many proteins and fats are made and transported. It is also the site for a lot of metabolic processes, and the main place in the cell where calcium ions are stored. Many proteins need calcium ions to work properly, including metabolic enzymes. In obesity, the endoplasmic reticulum becomes less able to store calcium ions.

A protein called STIM1 senses and regulates the levels of calcium ions in the endoplasmic reticulum. When calcium levels drop, STIM1 moves along the endoplasmic reticulum membrane towards the part that is next to the cell surface. Here, STIM1 joins up with a calcium channel called Orai1. The STIM1-Orai1 complex allows calcium ions to enter the cell and replenish its levels in the endoplasmic reticulum.

Arruda, Pers et al. have now asked if STIM1 is altered in obesity and, if so, whether it contributes to the endoplasmic reticulum’s inability to maintain proper calcium levels. High-resolution microscopy and biochemical techniques confirmed that STIM1 is indeed compromised in liver cells from obese mice. In these cells, STIM1 was found in unusual small clusters. It also could not move along the endoplasmic reticulum membrane when calcium levels dropped. As a result of these navigational errors, STIM1 failed to couple with Orai1, meaning less calcium could enter the cell. Further work identified that a small sugar molecule that is added onto STIM1 in obesity is behind its reduced ability to move accurately.

Arruda, Pers et al. next created mice that lacked STIM1 in their liver. These mice showed signs of metabolic abnormalities. Notably, when STIM1 levels were experimentally increased in obese mice, it restored calcium levels in the endoplasmic reticulum closer to normal, and improved metabolism too.

Thus, regulating calcium levels in the endoplasmic reticulum via proteins such as STIM1 is essential for maintaining a healthy metabolism. Interventions to correct calcium levels may have therapeutic promise to combat metabolic diseases.

The endoplasmic reticulum (ER) is a key cellular organelle coordinating a variety of essential processes such as protein synthesis and secretion, lipid biosynthesis, glucose metabolism and redox reactions. Additionally, ER is the main site of Ca²⁺ storage in the cell (Gardner et al., 2013; Hotamisligil, 2010). Structurally, ER comprises a complex network of membranes spread throughout the cytoplasm, which establishes physical and functional contacts with many other organelles including mitochondria, endosomes, lipid droplets and the plasma membrane (Lynes and Simmen, 2011; Phillips and Voeltz, 2016).

Given its critical role in the cell, the functionality of the ER is tightly monitored by proteins that can communicate stress signals to other proteins or compartments of the cell in order to restore ER function. The most well-known adaptive pathway in the ER is the unfolded protein response (UPR), which has the general goal of restoring ER function by enhancing ER folding capacity, decreasing protein translation and increasing protein degradation (Gardner et al., 2013; Hetz et al., 2015; Hotamisligil, 2010; Wang and Kaufman, 2016). Over the past decade it has been widely documented that chronic stress imposed by excess nutrients and energy leads to ER dysfunction and UPR activation in a variety of metabolic tissues in both mouse models and obese humans, which is associated with cellular stress, inflammation, and metabolic dysfunction (Boden et al., 2008; Gregor et al., 2009; Hotamisligil, 2010; Nakatani et al., 2005; Ozcan et al., 2004; Sharma et al., 2008; Wang and Kaufman, 2016). Alleviation of ER stress by chemical or molecular chaperones dramatically improves metabolic control and insulin sensitivity and reduces inflammation in mouse models of obesity (Fu et al., 2015; Kammoun et al., 2009; Ozawa et al., 2005; Ozcan et al., 2006) as well as in humans (Kars et al., 2010; Xiao et al., 2011).

More recently, it has become clear that Ca²⁺ storage in the ER is compromised in the setting of obesity and other metabolic diseases (Arruda and Hotamisligil, 2015; Ozcan and Tabas, 2016). Ca²⁺ in the ER is essential for chaperone-mediated protein folding and secretion, as well as for the function of metabolic enzymes. Ca²⁺ concentration in the ER is tightly regulated by coordinated action between SERCA, which pumps Ca²⁺ from the cytosol into the ER lumen, and the IP₃ receptor (IP3R) or Ryanodine receptor, which release Ca²⁺ from the ER into the cytosol (Clapham, 2007). In obesity, hepatic SERCA activity is compromised (Fu et al., 2011; Meikle and Summers, 2017; Rong et al., 2013) while the activity of the IP3R1 Ca²⁺ channel is increased (Arruda et al., 2014; Feriod et al., 2017; Wang et al., 2012). These alterations result in decreased ER Ca²⁺ content, loss of folding capacity, ER stress, inflammation, impaired insulin action and abnormal glucose metabolism (Arruda and Hotamisligil, 2015). Additionally, in this setting a ‘leaky’ ER contributes to both higher cytosolic Ca²⁺ and mitochondrial Ca²⁺ uptake, which has important implications for cytosolic Ca²⁺ signaling and mitochondrial dysfunction seen in metabolic diseases (Arruda et al., 2014; Feriod et al., 2017; Ozcan et al., 2013; Wang et al., 2012; Xiao et al., 2011). Accordingly, strategies to restore hepatic ER Ca²⁺ levels, such as overexpression of SERCA or suppression of IP3R, improve ER function and promote metabolic homeostasis in mouse models of obesity (Arruda et al., 2014; Feriod et al., 2017; Fu et al., 2011; Wang et al., 2012). In a similar fashion, administration of compounds that increase ER Ca²⁺ content and improve ER function, e.g. SERCA agonists (Kang et al., 2016), IP3R antagonists (Ozcan et al., 2012), or azoromide (Fu et al., 2015), improves metabolic health in obese mice.

In addition to SERCA and IP3Rs, Ca²⁺ homeostasis in the ER is sensed and regulated by a third major adaptive/homeostatic system orchestrated by the STIM-Orai complex. STIM proteins (STIM1 and STIM2) are found in the ER and sense luminal Ca²⁺ through N-terminal Ca²⁺ binding EF hand domain. In the resting state, STIM proteins are bound to Ca²⁺ and spread evenly throughout the ER membrane. Upon ER Ca²⁺ release, STIM forms oligomers (seen in confocal microscopy as puncta), which translocate to junctions between ER and plasma membrane (PM), where they couple with the PM channel protein Orai1. This coupling results in the import of Ca²⁺ from the extracellular compartment to the cytosol, providing spatial Ca²⁺ signals that then influence cellular signaling and promote Ca²⁺ replenishment into the ER lumen through SERCA, in the process known as store operated Ca²⁺ entry or SOCE (Feske, 2007; Prakriya and Lewis, 2015; Soboloff et al., 2012; Wu et al., 2007).

Given that obesity leads to impaired ER Ca²⁺ handling and this is a key mechanism associated with ER dysfunction in the obese condition, we examined whether alterations in SOCE system exist and contribute to the inability of ER to restore and or maintain Ca²⁺ levels. Here we show that STIM1 is modified and SOCE is defective in hepatocytes from obese mice due to the inability of STIM1 to translocate and couple with Orai1 at the PM. Hepatocytes lacking STIM1 displayed stress, increased glucose production and insulin resistance, whereas over-expression of STIM1 in the liver of obese mice significantly improved glucose intolerance. These findings support a critical role for STIM1-mediated SOCE in the maintenance of healthy Ca²⁺ balance in the ER, insulin action, and systemic glucose metabolism.

In order to determine the impact of obesity on SOCE in the liver, we isolated primary hepatocytes from lean wild-type (WT) and genetically obese (Lep^ob/ob) mice and measured Ca²⁺ influx through STIM/Orai1 using the ratiometric calcium dye, Fura-2AM (Poenie and Tsien, 1986). First, the cells were incubated in Ca²⁺-free medium, and ER Ca²⁺ store depletion was induced by the addition of the SERCA inhibitor thapsigargin (Tg). In agreement with earlier reports (Arruda et al., 2014) the initial rise in cytosolic Ca²⁺ induced by Tg, which reflects the ER Ca²⁺ content, was significantly lower in hepatocytes isolated from obese animals compared with WT cells (Figure 1A). Next, we induced SOCE through STIM/Orai1 by substituting the Ca²⁺-free media with a media containing 2 mM Ca²⁺. As depicted in Figure 1A, the Ca²⁺ entry in primary hepatocytes from obese mice was markedly reduced relative to control cells. To further confirm that the observed Ca²⁺ entry was mediated by SOCE, we added the SOCE inhibitor 2-apb, which completely blocked Ca²⁺ influx in both genotypes (Figure 1A, dotted lines). This finding is consistent with a report of impaired SOCE in the steatotic hepatocytes from Zucker rats (Wilson et al., 2015).

Figure 1 with 3 supplements see all

Download asset Open asset

Figure 1—source data 1

Source data for Figure 1.

https://doi.org/10.7554/eLife.29968.004

Download elife-29968-fig1-data1-v2.xlsx

As we observed impaired SOCE in hepatocytes from obese animals we next evaluated whether the expression levels of the main SOCE components were altered in obesity. As shown in Figure 1—figure supplement 1A, we did not detect differences in expression of Orai1 in the livers of mice with genetic or high-fat diet (HFD)-induced obesity. Despite a modest increase in the mRNA levels of stim1 and stim2, protein levels of STIM1, STIM2 and Orai1 remained unchanged in the livers of mice with both genetic and diet induced obesity (Figure 1B and Figure 1—figure supplement 1B). However, phospho-JNK (pJNK), a marker of inflammatory stress (Hirosumi et al., 2002) and phospho-Calmodulin kinase (pCaMKII), a marker of elevated cytosolic Ca²⁺ (Ozcan et al., 2012), were increased in the livers of obese animals (Figure 1B and Figure 1—figure supplement 1B). Thus decreased Ca²⁺ import through SOCE in hepatocytes derived from obese mice appears to be independent of the expression levels of critical SOCE components.

We therefore asked whether the decreased Ca²⁺ import through SOCE in hepatocytes from obese mice was a result of a functional alteration in STIM1 translocation in the ER membrane. To evaluate the activity of STIM1 proteins in obesity, we first validated antibodies for endogenous immunostaining (Figure 1; Figure 1—figure supplement 1C). Next, we isolated primary hepatocytes from lean and obese mice, induced Ca²⁺ store depletion with Tg and determined STIM1 localization by immunostaining. As shown in Figure 1C and D and Figure 1—figure supplement 1D, while STIM1 was evenly distributed in the ER membrane of resting cells, Tg treatment led to a dramatic translocation to areas of the ER membrane in close proximity with the PM. This effect was observed within 5–10 min of Tg treatment and persisted for 30 min. However, in cells from obese (Lep^ob/ob) mice, STIM1 distribution at baseline was punctate throughout the entire ER (Figure 1D and quantified in Figure 1E), and its translocation to areas of ER/PM junction in response to Tg was dramatically impaired. As shown in Figure 1F and Figure 1—figure supplement 1E, in WT cells, Tg treatment increased the number of high-intensity pixels, indicating the formation of the punctate structures, which represent oligomerization of STIM proteins. In contrast, hepatocytes derived from obese mice displayed higher intensity pixel counts at baseline with no further increase following Tg treatment. Defective STIM1 translocation in hepatocytes from obese animals is also illustrated in the line graphs showing the representative intensity profile of individual cells before and after Tg treatment (Figure 1G and also see Figure 1—figure supplement 1F for image analysis details). In WT cells, Tg treatment increased the signal intensity at the edge of the cell and decreased it in the cytosol, quantified as the STIM1 ratio between these compartments (Figure 1H). In hepatocytes from obese mice, the average signal intensity at baseline was higher than in WT cells, and did not change significantly in response to Tg (Figure 1H), indicating that STIM1 translocation is markedly defective in these cells.

To examine STIM1 translocation after Tg stimulation in more detail and using an alternative analytical tool, we performed total internal reflection fluorescence (TIRF) microscopy, in which fluorophores in the vicinity of the cell surface are selectively excited. As shown in Figure 1I, in WT cells Tg treatment induced STIM1 puncta formation and accumulation in areas close to the PM. This response was diminished in cells derived from obese animals (Figure 1J). In these experiments, Na⁺K⁺ ATPase was used as a constitutive marker of PM and its staining pattern did not change in the presence of Tg.

We also evaluated the time course of the defect in STIM1 trafficking during the development of obesity in mice. As shown in Figure 1—figure supplement 2A and Figure 1—figure supplement 2B, after 3 and 5 weeks of HFD, STIM1 showed some degree of translocation towards the plasma membrane in resting cells, indicating that short term HFD exposure is sufficient to trigger ER Ca²⁺ release, the driving force for STIM1 translocation. Additionally, at 3 weeks HFD, STIM1 translocation stimulated by Tg was not significantly impaired compared with cells derived from chow fed animals. However, the defect in STIM1 translocation induced by Tg was apparent in cells isolated from mice fed a HFD for 5 weeks and was more pronounced after 7 and 11 weeks of HFD (Figure 1—figure supplement 2A and Figure 1—figure supplement 2B). These data support that defective STIM1 translocation may be a common feature of obesity in independent experimental models (Lep^ob/ob and HFD) and that this phenotype occurs independent of blood glucose levels and prior to the emergence of marked hyper-insulinemia and insulin resistance found in obesity (Figure 1—figure supplement 2C).

Next, we examined the activation and translocation of STIM2 in cells from lean (WT) and obese (Lep^ob/ob) mice. The role of STIM2 in the regulation of SOCE has been explored only recently, and its activation seems to result from smaller fluctuations in Ca²⁺ due to its lower affinity for the ion, while STIM1 is activated when the extent of ER depletion increases (Berna-Erro et al., 2017; Oh-Hora et al., 2008; Prakriya and Lewis, 2015). In hepatocytes, STIM2 is expressed at a lower level than STIM1 (López et al., 2012; Williams et al., 2001), however it was detectable both by staining and by western blot analysis. We did not observe aberrant STIM2 puncta formation in cells from Lep^ob/ob mice but a tendency for translocation at baseline. Upon Tg-induced store depletion, we observed a marked STIM2 translocation to areas close to the PM in both WT cells and Lep^ob/ob cells, however the degree of translocation in Lep^ob/ob cells was less pronounced than in WT cells (Figure 1—figure supplement 3A). Thus, the obesity-related defect in STIM2 translocation was present but less dramatic than that observed for STIM1. Overall, these findings suggest that defective SOCE in primary hepatocytes from obese mice is predominantly related to aberrant STIM1 puncta formation and inefficient STIM1 re-localization from the bulk ER membrane to ER/PM junction areas after ER Ca2+ store depletion.

In order to gain insight into mechanisms underlying this phenomenon, we considered the possibility that altered STIM1 post-translational modification could be involved in its defective translocation capacity in the context of obesity. Two known post-translational modifications of STIM1 which influence its trafficking are phosphorylation and O-GlcNAcylation (Pozo-Guisado et al., 2013; Zhu-Mauldin et al., 2012). Phosphorylation of STIM1 at Ser621 and Ser575 regulate its interaction with the microtubule plus-end-tracking protein EB1, enabling STIM1 to move in the ER membrane (Pozo-Guisado et al., 2013). Therefore, we asked whether a reduction in STIM1 phosphorylation at these sites may explain its defective trafficking in obesity. Surprisingly however, we found that phosphorylation of STIM1 at Ser621 and Ser575 was actually increased in liver lysates from obese mice (Figure 2A). This indicates that lack of phosphorylation at these residues does not underlie the defective translocation of STIM1, and suggests that hepatic STIM1 may be released from EB1 in the basal state, potentially as a response to the decreased ER Ca²⁺ level observed in hepatocytes from obese mice.

Figure 2 with 1 supplement see all

Download asset Open asset

STIM1 can also be modified by O-linked N-acetyl glucosamine (O-GlcNAc), a post-translational modification of serine or threonine amino acids. Previous work has shown that STIM1 O-GlcNAcylation impairs the ability of the protein to move in the ER membrane and to form punctate structures (Zhu-Mauldin et al., 2012). Additionally, conditions of nutrient and substrate excess, including obesity, lead to increase in cellular O-GlcNAcylation levels (Vosseller et al., 2002; Yang and Qian, 2017; Dentin et al., 2008; Yang et al., 2008). These studies indicate the possibility that metabolic stress may interfere with SOCE via O-GlcNAcylation of STIM1, which disrupts its proper trafficking. To test this hypothesis, we first examined global O-GlcNAcylation in primary hepatocytes from WT and obese mice. As shown in Figure 2B, and in agreement with previous observations (Baldini et al., 2016), global O-GlcNAcylation was higher in hepatocytes derived from obese animals compared with their controls. This effect was amplified by treatment with PugNac, a specific inhibitor of O-GlycNAcase (OGA), the enzyme that catalyzes removal of OglcNAc sugars from proteins. Next, we performed immunoprecipitation of O-GlcNAc-modified proteins using an OglcNAc-specific antibody from hepatocyte lysates. We found that STIM1 was present among the OglcNAcylated proteins and it was more abundant in cells from obese mice (Figure 2C). As a complementary approach, we utilized biotinylated-succinylated wheat germ agglutinin (succinylated-WGA), a lectin that preferentially binds N-acetylglucosamine versus other sugars (Baldini et al., 2016; Hu et al., 2010). Following precipitation with streptavidin-conjugated magnetic beads, STIM1 was detected in the pool of proteins modified by O-GlcNac at higher levels in samples derived from obese animals relative to WT controls (Figure 2D). Additionally, as shown in Figure 2E, OGT precipitates with STIM1 in hepatocytes derived from obese animals, indicating that OGT and STIM1 exist in a complex in obesity, consistent with higher levels of O-GlcNac-modified STIM1 in this condition. To explore whether STIM1 is also modulated by OglcNAcylation in the HFD context and study the time course of this modification, we isolated hepatocytes from animals fed a HFD for 3, 5 and 11 wks and pull down STIM1 using succinylated-WGA. As shown in Figure 2—figure supplement 1A, STIM1 was progressively modified by O-GlcNac in this context starting at 3 weeks on HFD.

In order to test if O-GlcNAc modification may alter STIM1 function in hepatocytes, we first overexpressed OGT in Hepa1-6 cells. As expected, overexpression of OGT strongly increased global protein modification with O-GlcNac (Figure 2—figure supplement 1B). We then co-expressed OGT and STIM1 tagged with a Flag peptide (Figure 2—figure supplement 1C) to determine whether STIM1 can be directly O-GlcNacylated by OGT in this system. Immunoprecipitation of STIM1-Flag with a Flag specific antibody demonstrated its enhanced O-GlcNAc modification in OGT overexpressing cells (Figure 2F). Additionally, STIM1 is able to directly bind to OGT in this cell model (Figure 2F), similar to what we observed in hepatocytes derived from obese animals (Figure 2E). Having established this cellular system with enhanced STIM1 O-GlcNacylation, we then assessed its translocation in cells transfected with OGT labeled with RFP, using un-transfected cells in the same culture plate as internal controls for quantification (Figure 2G). These experiments showed that STIM1 puncta formation and translocation following Tg treatment was markedly impaired in cells overexpressing OGT compared with control cells (Figure 2G,H and I). Additionally, overexpression of OGT led to a significant impairment in SOCE compared with the controls (Figure 2J), with no significant differences in the response to Tg (data not shown).

Next, to determine whether inhibition of the O-GlcNac modification could rescue STIM1 translocation defect in Lep^ob/ob cells, we used adenoviral gene delivery to express an shRNA targeting OGT to down-regulate OGT and thus the O-GlcNacylation capacity of the cell. As can be seen in Figure 2K and L, the expression of OGT shRNA lead to a 60% decrease in its expression and activity. As shown in Figure 2M and N, down-regulation of OGT resulted in increased STIM1 translocation capacity compared with cells expressing a scrambled shRNA. Taken together these data indicate that increased modification of STIM1 by OglcNAc could, at least in part, underlie defective STIM1 translocation and reduced hepatic SOCE in the context of obesity.

We then started to investigate the impact of defective STIM1-mediated SOCE on ER homeostasis, cellular stress responses and metabolic regulation in STIM1-deficient hepatocyte cell models. These included primary hepatocytes derived from mice with genetic STIM1 deficiency specifically in the liver (stim1^fl/fl Alb;Cre, identified here as stim1Δ^LIVER) and Hepa1-6 cells stably expressing shRNAs targeting stim1 (Figure 3—figure supplement 1A) or stim2 (Figure 3—figure supplement 1B) (identified here as shSTIM1 and shSTIM2 respectively). Primary hepatocytes derived from stim1Δ^LIVER mice displayed lower ER Ca²⁺ content and absence of Tg-triggered SOCE compared to controls (stim1^fl/fl) (Figure 3A). Similarly, Hepa 1–6 cells in which STIM1 expression was down regulated showed markedly blunted SOCE (Figure 3—figure supplement 1C). Imbalances in ER Ca²⁺ content trigger cellular stress responses through various pathways (Arruda and Hotamisligil, 2015; Fu et al., 2012; Ozcan and Tabas, 2016). In order to examine these responses in our cellular models of STIM deficiency, we measured phosphorylation of JNK as a benchmark measure of cellular stress and inflammatory activation, and phosphorylation of eIF2α as a marker of UPR activation. As shown in Figure 3B, in stim1^fl/fl control cells, treatment with Tg induced phosphorylation of JNK and eIF2α at 30 min followed by a decrease in phosphorylation levels back to baseline after 90 min. However, in STIM1-deficient cells, the phosphorylation of JNK and eIF2α was enhanced at baseline and these cells displayed a stronger and more persistent response to Tg, with stress markers only returning to basal levels at 120 min. A similar profile was observed in Hepa1-6 cells with shRNA-mediated suppression of STIM1 (Figure 3—figure supplement 1D). Interestingly, suppression of STIM2 alone did not alter the cellular response to Tg (Figure 3—figure supplement 1E). Altogether, these data demonstrate that absence of core components of SOCE leads to elevated and prolonged stress responses in hepatocytes.

Figure 3 with 2 supplements see all

Download asset Open asset

Figure 3—source data 1

Source data for Figure 3.

https://doi.org/10.7554/eLife.29968.014

Download elife-29968-fig3-data1-v2.xlsx

Increased cellular stress and inflammation are associated with impaired insulin action and defective glucose and lipid metabolism (Fu et al., 2012; Hirosumi et al., 2002; Hotamisligil, 2017). Accordingly, we found that primary hepatocytes derived from stim1Δ^LIVER mice displayed impaired phosphorylation of IRβ and AKT in response to insulin relative to cells from stim1^fl/fl littermates (Figure 3C). Likewise, gene silencing of stim1 in Hepa1-6 cells resulted in decreased insulin signaling (Figure 3—figure supplement 1F). Additionally, primary hepatocytes isolated from stim1Δ^LIVER mice maintained on HFD showed higher levels of glucose production stimulated by the gluconeogenesis substrates glycerol, pyruvate and glutamine compared to stim1^fl/fl derived hepatocytes (Figure 3E). In agreement with our finding that STIM2 suppression does not amplify stress responses, we found that STIM2-deficient cells retained normal insulin responsiveness (Figure 3—figure supplement 1G).

Recently, it has been shown that SOCE may regulate lipid metabolism (Maus et al., 2017; Wilson et al., 2015) and animals with inducible STIM1/2 whole body deficiency exhibit increased lipid accumulation in multiple tissues such as skeletal muscle, heart and liver, as a consequence of impaired lipolysis and fatty acid oxidation (Maus et al., 2017). We observed that primary hepatocytes isolated from stim1Δ^LIVER mice showed modestly increased accumulation of lipid droplets at baseline and after incubation with 1 mM oleic acid and 40 µM of palmitic acid (Figure 3D). These results suggest that in addition to higher stress levels, insulin resistance and increased glucose production, SOCE deficiency may result in abnormal lipid metabolism.

We next assessed the systemic metabolic impact of STIM1 deficiency in hepatocytes in vivo in the stim1Δ^LIVER mice described above. As shown in Figure 3—figure supplement 2A, liver lysates from stim1Δ^LIVER mice displayed marked reduction in STIM1 and a mild increase in STIM2 protein. On a chow diet, stim1Δ^LIVER mice gained weight equivalently to stim1^fl/fl mice (Figure 3—figure supplement 2B) and did not exhibit alterations in glucose tolerance and triglyceride content (Tg) (data not shown). In the HFD-fed mice, the weight gain was similar between genotypes (Figure 3—figure supplement 2B). Interestingly, at 6 weeks on a HFD, we observed a mild, but significant, glucose intolerance in the stim1Δ^LIVER animals compared with the stim1^fl/fl controls (Figure 3—figure supplement 2C). Additionally, at this time point, insulin sensitivity was impaired (Figure 3—figure supplement 2D). No significant differences were observed in Tg content between the two genotypes, although a tendency to higher Tg levels was detected in stim1Δ^LIVER mice (Figure 3—figure supplement 2E). In long term HFD (20 weeks), the differences in glucose intolerance, insulin signaling and Tg content between stim1^fl/fl and stim1Δ^LIVER groups were very mild and did not reach statistical significance (Figure 3—figure supplement 2F,G and H).

Interestingly, although we haven’t observed overall changes in lipid accumulation in the liver specific STIM1 deficient animals, we have noticed that these animals showed higher content of microvesicular steatosis compared with controls where the majority of the lipid droplets are presented as large droplets (Figure 3—figure supplement 2I).

In light of the mild in vivo metabolic phenotype of constitutive hepatocyte-specific STIM1 deletion during development, we considered that STIM2 upregulation (Figure 3—figure supplement 2) or alternative systems could compensate for the lack of STIM1 all throughout the embryonic life. Therefore, we examined the effect of acute deletion of STIM1 in hepatocytes, using adenovirus mediated gene delivery to express albumin-Cre recombinase (Alb;Cre) in adult stim1^+/+ and stim1^fl/fl mice. After weaning, littermate animals were subjected to HFD for 4 weeks (for a short-term stress induction) (Figure 3F). Adenoviral delivery of Alb;Cre recombinase resulted in ~60% deletion of hepatic STIM1 (Figure 3G). Importantly, there was no compensatory upregulation of STIM2 or Orai1 in this setting. Indeed, one week after adenovirus administration, the acute deletion of STIM1 resulted in significantly higher fasting glucose levels (Figure 3H) without any difference in insulin levels (Figure 3I) and body weight (data not shown). Interestingly, following STIM1 deletion, mice also exhibited significantly impaired glucose tolerance (Figure 3J). In summary, acute SOCE dysfunction in the liver results in amplified cellular stress and glucose intolerance in mice fed a HFD.

Based on these observations, and the marked SOCE defect associated with obesity we hypothesized that recovery of SOCE function in the livers of obese mice could improve metabolic homeostasis in obesity. To test this hypothesis, we used a hepatocyte-specific adenovirus system to exogenously express either GFP (control) or STIM1-YFP in the Lep^ob/ob primary hepatocytes and mice (Figure 4A). The functionality of the STIM1-YFP fusion protein has been verified previously (Liou et al., 2005) and confirmed by us (Video 1).

Figure 4 with 1 supplement see all

Download asset Open asset

Figure 4—source data 1

Source data for Figure 4.

https://doi.org/10.7554/eLife.29968.020

Download elife-29968-fig4-data1-v2.xls

Video 1

Download asset

Using this approach, we asked whether exogenous expression of STIM1-YFP would be sufficient to overcome the SOCE defects in primary hepatocytes from obese animals. As shown in Figure 4B, in control Lep^ob/ob cells expressing GFP, STIM1 formed puncta at baseline and Tg-induced translocation of the protein was impaired, as we previously observed (Figure 1D). Delivery of STIM1-YFP lead to a significant increase in the protein level in liver cells. Interestingly, in non treated cells, we observed that some degree of the STIM1 protein was already localized in areas of close contact with the plasma membrane even prior to stimulation (Figure 4C). Additionally, exogenously expressed STIM1 was able to translocate towards the plasma membrane after Tg treatment (Figure 4C and D). To examine STIM1 translocation in Lep^ob/ob hepatocytes with higher resolution, we also performed TEM in cells expressing exogenous STIM1 fused with HRP. STIM1-HRP-expressing cells were fixed and treated with diaminobenzidine (DAB) in the presence of H₂O₂. HRP catalyzes the polymerization and deposition of DAB, which recruits electron-dense osmium, providing contrast and revealing the localization of STIM1. As shown in Figure 4E, exogenous delivery of STIM1-HRP resulted in rescue of translocation in cells from obese animals in a manner similar to that observed in lean, wild type cells. Taken together, these data indicate that exogenously increasing the amount of STIM1 protein is sufficient to partially overcome the STIM1 translocation defect in cells from obese mice.

The ability of STIM1-YFP overexpression to overcome, at least in part, the translocation defect of endogenous STIM1 in Lep^ob/ob cells suggests that at least some proportion of STIM1 in this setting may escape the O-GlycNac modification. To examine the degree of O-GlcNacylation of overexpressed STIM1-YFP in Lep^ob/ob cells, we used an O-GlcNac specific antibody to immunoprecipitate all proteins modified by O-GlycNacylation in GFP or STIM1 YFP overexpressing cells and examined STIM1 protein by immunoblotting. As shown in Figure 4F, STIM1-YFP overexpression resulted in an ~20 fold increase in the expression of STIM1 protein. However, the amount of O-GlcNac modified STIM1 did not increase proportionally, indicating that in fact a significant amount of the overexpressed STIM1 escapes this post translation modification.

Additionally, it has been previously shown that overexpression of STIM1 in HeLa cells leads to morphological changes in the ER with an increased cortical ER (Orci et al., 2009). In agreement with this finding, electron micrographs of primary hepatocytes derived from Lep^ob/ob mice show that STIM1-YFP expression led to a remodeling of ER with abundant ER stacks apposed to the PM, although the degree of ER remodeling varied from cell to cell, likely due to variable STIM1 expression levels (Figure 4—figure supplement 1A). This result suggests that, in addition to having more STIM1 that translocates to PM, overexpression of STIM1-YFP also remodels the ER, favoring the proximity of STIM1 to Orai at the plasma membrane.

After verifying that overexpression of STIM1-YFP was able to rescue STIM1 translocation defects in obese cells, we examined whether this actually resulted in improved SOCE. As shown in Figure 4G overexpression of STIM1-YFP in Lep^ob/ob cells resulted in increased ER Ca²⁺ and SOCE compared to control cells. Thus, overexpression of STIM1 was a successful intervention to overcome the STIM translocation and SOCE defects, resulting in improved ER calcium handling.

Next, we asked whether the effects of the overexpression of STIM1-YFP in Lep^ob/ob primary hepatocytes would impact overall metabolism in vivo, and evaluated the effect of liver-specific STIM1 expression on systemic glucose metabolism in the Lep^ob/ob mice following the protocol displayed in Figure 5A. Introduction of STIM1 by adenovirus gene delivery led to increased STIM1 protein levels in the liver. Interestingly, increased STIM1 expression was also accompanied by increased mRNA expression of Orai1 and with an increase in SERCA (Atp2a2) mRNA and protein levels (Figure 5B and C). This is in accordance with previous work showing that the modulation of expression STIM1 affects the expression of other Ca²⁺ channels or pumps, as SERCA, likely due to alterations in cytosolic Ca²⁺ levels (Abell et al., 2011). Remarkably, liver-specific overexpression of STIM1 led to a significant improvement in glucose tolerance relative to control (adGFP) animals as evaluated by a glucose tolerance test (Figure 5D). Improved glucose tolerance resulting from overexpression of STIM1 was associated with enhanced in vivo insulin signaling evaluated by direct insulin injection into the livers that resulted in increased AKT phosphorylation (Figure 5E). Additionally, expression of genes involved in glycolysis were increased in livers overexpressing STIM1, accompanied by decreased levels of PCK1 (PEPCK), a rate limiting enzyme for gluconeogenesis (Figure 5F and G).

Figure 5 with 1 supplement see all

Download asset Open asset

Figure 5—source data 1

Source data for Figure 5.

https://doi.org/10.7554/eLife.29968.024

Download elife-29968-fig5-data1-v2.xlsx

Given the reported role of STIM1 in lipid metabolism, we also evaluated if overexpression of STIM1 impacted the excessive lipid accumulation in the livers of Lep^ob/ob animals. As shown in Figure 5H, exogenous expression of STIM1 led to a reduction in total Tg content in the liver tissue of Lep^ob/ob mice compared to controls expressing GFP. Accordingly, STIM1 overexpression led to increase mRNA expression of genes involved in fatty acid oxidation, such as CPT1, while no change was observed in genes involved in lipogenesis (Figure 5—figure supplement 1A and B). Based on these results we conclude that SOCE is critical for glucose and lipid metabolism and that increasing hepatic SOCE through overexpression of STIM1 is able to revert, at least in part, the deleterious impact of obesity on systemic glucose homeostasis.

In the last several years, a growing number of studies have laid the foundation for the concept that disrupted intracellular Ca²⁺ homeostasis in metabolic tissues is a key component of ER dysfunction and metabolic deterioration (Arruda and Hotamisligil, 2015; Ozcan and Tabas, 2016). The abnormal Ca²⁺ handling in the ER of hepatocytes in this setting is multi-faceted, involving both impairment of SERCA activity (Fu et al., 2011) as well as increased activity of IP3R (Arruda et al., 2014; Feriod et al., 2017; Wang et al., 2012). This impacts not only Ca²⁺ levels in the ER and protein folding, but also mitochondrial oxidation and ROS production as well as the activation of cytosolic stress signaling pathways. Supporting the critical role of this biology, human genetic studies have shown that SNPs in SERCA (Varadi et al., 1999) and IP3R (Shungin et al., 2015) are associated with metabolic diseases such as obesity and diabetes. Here, our work reveals that another key mechanism supporting ER Ca²⁺ homeostasis, STIM-mediated SOCE, is also defective in obesity with important implications for the metabolic dysfunction in hepatocytes. Strikingly, we demonstrate that STIM1 gain-of-function improves cellular stress responses and glucose homeostasis in obese mice, underscoring the therapeutic potential of targeting Ca²⁺ homeostasis through STIM1 for metabolic disease treatment.

Our interest in this biology was heightened by our initial observation that in hepatocytes isolated from obese (Lep^ob/ob) mice, STIM1 was present in an aberrant punctate pattern along the ER membrane. One possible explanation was that Lep^ob/ob hepatocytes display increased SOCE even in baseline conditions in order to respond to low ER Ca²⁺ levels induced by dysfunction of SERCA and IP3R1. However, our detailed examination revealed that in cells of obese mice, STIM1 puncta were not localized in areas of close proximity of the PM, but rather were distributed all throughout the cell away from the PM. Additionally, when we depleted ER Ca²⁺ stores, STIM1 was not able to fully translocate to the PM, resulting in significantly reduced SOCE. Hence, metabolic stress impairs this critical component of ER Ca²⁺ homeostasis in obese liver tissue.

One question arising from these observations is the nature of the mechanism underlying STIM1 dysfunction in obesity. STIM activation and translocation is modulated by a complex array of factors that include post-translational modification of the protein itself, protein-protein interactions (for review Lopez et al., 2016), the interaction of STIM1 with lipids of the ER membrane and the ability of the ER itself to remodel and form ER/PM junctions (Derler et al., 2016; Lopez et al., 2016; Prakriya and Lewis, 2015). Here we found that in hepatocytes derived from Lep^ob/ob mice, STIM1 displays significantly increased levels of O-GlcNAcylation, a post-translation modification that causes impaired STIM1 trafficking and function (Zhu-Mauldin et al., 2012). Indeed, overexpression of OGT in hepatocytes was sufficient to induce abnormal STIM1 puncta formation and impaired activation of SOCE while down-regulation of OGT in primary hepatocytes from obese mice partially rescued the STIM1 translocation defect. It will be interesting to explore the detailed location and impact of these modifications in future studies. In silico analysis, published literature and data in dbOGAP, suggest potential target sites coinciding with the domains involved in STIM1/Orai1 interactions with Orai1 in the CAD/SOAR domain or in the polybasic motif located at the C terminal of the protein that may be critical in oligomerization and puncta formation (Liou et al., 2007). Regardless, protein modification with O-GlcNAc is a reflection of the nutritional status, as it integrates glucose, amino acid, fatty acid and nucleotide flux through the hexosamine biosynthetic pathway (HBP) to produce N-acetyl-glucosamine (UDP-GlcNAc), the obligatory substrate for OGT. Hence, conditions of nutrient and substrate excess, including obesity, lead to increased cellular O-GlcNAcylation levels (Vosseller et al., 2002; Yang and Qian, 2017; Dentin et al., 2008; Yang et al., 2008). Taken together, our findings demonstrate that O-GlcNAc modification of STIM1 alters organelle Ca²⁺ regulation with consequences for ER function and systemic metabolism and provide an important mechanistic insight into regulation of this critical metabolic integration point through organelle homeostasis in obesity.

Another important finding reported here is that impaired SOCE in hepatocytes by STIM1 deficiency was sufficient to induce markers of ER stress and inflammation, to impair of insulin action, increase glucose production and induce excessive lipid droplet accumulation. Recently, it has been reported that cells from patients with loss-of-function mutations in STIM1 or ORAI accumulate lipid droplets as a consequence of their inability to increase cAMP, mobilize fatty acids from lipid droplets, activate lipolysis, and oxidize fatty acids (Maus et al., 2017). Interestingly, tamoxifen-induced whole body STIM1/2 deficiency leads to higher lipid content in the heart, muscle and liver (Maus et al., 2017). Here, although we detected increased accumulation of lipid droplets in hepatocytes lacking STIM1 in vitro, these phenomena were not clearly observed in vivo. Hepatocyte-specific deletion of STIM1 led to a mild metabolic phenotype in vivo suggesting that the activation of compensatory systems occurs when STIM1 is deleted during development. Indeed, STIM2 expression levels were slightly increased in STIM1 deficient liver. Furthermore, in lean animals, the rest of the ER calcium handling systems are intact, which may allow maintenance of the normal equilibrium. In contrast, acute down-regulation of STIM1 showed a more marked effect on glucose homeostasis.

Finally, we show here that exogenous expression of STIM1 in primary hepatocytes from obese mice is sufficient to overcome SOCE defects and partially correct ER Ca²⁺ levels and STIM1 re-localization upon Ca²⁺ store depletion. Notably, we showed that a significant part of the exogenously expressed STIM1 is able to escape the O-GlcNAc modification, probably by a mass effect, and responds properly to ER Ca²⁺ depletion. Additionally, overexpression of STIM1 in hepatocytes from Lep^ob/ob animals led to remodeling of the ER with an increase in the amount of cortical ER possibly facilitating STIM1 translocation and Orai1 coupling. Indeed, it is known that STIM1 regulates the structure of the ER through its role in Tip Attachment Complex movement (Grigoriev et al., 2008; Westrate et al., 2015), coordinating the growth and shrinkage of the ER tubule. Importantly, we show that upregulation of SOCE through STIM1 replenishment in the liver of obese mice results in significant metabolic benefit including improved insulin signaling and glucose tolerance and decreased lipid content likely as a result of enhanced fatty acid oxidation. When STIM1 is exogenously supplied to the liver tissue in obese mice, we also observed an increase in glycolytic gene expression. This is in agreement with recent report showing that in T cells SOCE regulates the expression of glycolytic genes through the modulation of the transcription factor NFAT (Vaeth et al., 2017). Overexpression of STIM1 also led to increased SERCA levels, which is necessary for the delivery of Ca²⁺ into the ER. In fact, it is proposed and validated experimentally and computationally that an adaptive feedback loop between components of Ca²⁺ machinery exist to keep cytosolic Ca²⁺ levels under control (Abell et al., 2011). Increased SERCA expression in the context of STIM1 overexpression may indeed be a critical component of the metabolic benefit resulting from STIM1 expression in the liver in obese mice. It is remarkable that the individual manipulation of multiple proteins involved in the maintenance of ER Ca²⁺ levels- SERCA, IP3R, and STIM1- all result in a similar phenotype. These findings underscore the importance of ER Ca²⁺ homeostasis and ER function to metabolic health, and indicate the strong potential for targeting these pathways to combat metabolic disease.

1. 1. Abell E
   2. Ahrends R
   3. Bandara S
   4. Park BO
   5. Teruel MN

   (2011) Parallel adaptive feedback enhances reliability of the Ca2+ signaling system

   PNAS 108:14485–14490.

   https://doi.org/10.1073/pnas.1018266108

   • PubMed
   • Google Scholar
2. 1. Arruda AP
   2. Pers BM
   3. Parlakgül G
   4. Güney E
   5. Inouye K
   6. Hotamisligil GS

   (2014) Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity

   Nature Medicine 20:1427–1435.

   https://doi.org/10.1038/nm.3735

   • PubMed
   • Google Scholar
3. 1. Arruda AP
   2. Hotamisligil GS

   (2015) Calcium Homeostasis and Organelle Function in the Pathogenesis of Obesity and Diabetes

   Cell Metabolism 22:381–397.

   https://doi.org/10.1016/j.cmet.2015.06.010

   • PubMed
   • Google Scholar
4. 1. Baldini SF
   2. Wavelet C
   3. Hainault I
   4. Guinez C
   5. Lefebvre T

   (2016) The Nutrient-Dependent O-GlcNAc Modification Controls the Expression of Liver Fatty Acid Synthase

   Journal of Molecular Biology 428:3295–3304.

   https://doi.org/10.1016/j.jmb.2016.04.035

   • PubMed
   • Google Scholar
5. 1. Berna-Erro A
   2. Jardin I
   3. Salido GM
   4. Rosado JA

   (2017) Role of STIM2 in cell function and physiopathology

   The Journal of Physiology 595:3111–3128.

   https://doi.org/10.1113/JP273889

   • PubMed
   • Google Scholar
6. 1. Boden G
   2. Duan X
   3. Homko C
   4. Molina EJ
   5. Song W
   6. Perez O
   7. Cheung P
   8. Merali S

   (2008) Increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals

   Diabetes 57:2438–2444.

   https://doi.org/10.2337/db08-0604

   • PubMed
   • Google Scholar
7. 1. Clapham DE

   (2007) Calcium signaling

   Cell 131:1047–1058.

   https://doi.org/10.1016/j.cell.2007.11.028

   • PubMed
   • Google Scholar
8. 1. Dentin R
   2. Hedrick S
   3. Xie J
   4. Yates J
   5. Montminy M

   (2008) Hepatic glucose sensing via the CREB coactivator CRTC2

   Science 319:1402–1405.

   https://doi.org/10.1126/science.1151363

   • PubMed
   • Google Scholar
9. 1. Derler I
   2. Jardin I
   3. Romanin C

   (2016) Molecular mechanisms of STIM/Orai communication

   American Journal of Physiology-Cell Physiology 310:C643–C662.

   https://doi.org/10.1152/ajpcell.00007.2016

   • PubMed
   • Google Scholar
10. 1. Feriod CN
    2. Oliveira AG
    3. Guerra MT
    4. Nguyen L
    5. Richards KM
    6. Jurczak MJ
    7. Ruan HB
    8. Camporez JP
    9. Yang X
    10. Shulman GI
    11. Bennett AM
    12. Nathanson MH
    13. Ehrlich BE

    (2017) Hepatic Inositol 1,4,5 Trisphosphate Receptor Type 1 Mediates Fatty Liver

    Hepatology Communications 1:23–35.

    https://doi.org/10.1002/hep4.1012

    • PubMed
    • Google Scholar
11. 1. Feske S

    (2007) Calcium signalling in lymphocyte activation and disease

    Nature Reviews Immunology 7:690–702.

    https://doi.org/10.1038/nri2152

    • PubMed
    • Google Scholar
12. 1. Fu S
    2. Yang L
    3. Li P
    4. Hofmann O
    5. Dicker L
    6. Hide W
    7. Lin X
    8. Watkins SM
    9. Ivanov AR
    10. Hotamisligil GS

    (2011) Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity

    Nature 473:528–531.

    https://doi.org/10.1038/nature09968

    • PubMed
    • Google Scholar
13. 1. Fu S
    2. Watkins SM
    3. Hotamisligil GS

    (2012) The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling

    Cell Metabolism 15:623–634.

    https://doi.org/10.1016/j.cmet.2012.03.007

    • PubMed
    • Google Scholar
14. 1. Fu S
    2. Yalcin A
    3. Lee GY
    4. Li P
    5. Fan J
    6. Arruda AP
    7. Pers BM
    8. Yilmaz M
    9. Eguchi K
    10. Hotamisligil GS

    (2015) Phenotypic assays identify azoramide as a small-molecule modulator of the unfolded protein response with antidiabetic activity

    Science Translational Medicine 7:292ra98.

    https://doi.org/10.1126/scitranslmed.aaa9134

    • Google Scholar
15. 1. Gardner BM
    2. Pincus D
    3. Gotthardt K
    4. Gallagher CM
    5. Walter P

    (2013) Endoplasmic reticulum stress sensing in the unfolded protein response

    Cold Spring Harbor Perspectives in Biology 5:a013169.

    https://doi.org/10.1101/cshperspect.a013169

    • PubMed
    • Google Scholar
16. 1. Gregor MF
    2. Yang L
    3. Fabbrini E
    4. Mohammed BS
    5. Eagon JC
    6. Hotamisligil GS
    7. Klein S

    (2009) Endoplasmic reticulum stress is reduced in tissues of obese subjects after weight loss

    Diabetes 58:693–700.

    https://doi.org/10.2337/db08-1220

    • PubMed
    • Google Scholar
17. 1. Grigoriev I
    2. Gouveia SM
    3. van der Vaart B
    4. Demmers J
    5. Smyth JT
    6. Honnappa S
    7. Splinter D
    8. Steinmetz MO
    9. Putney JW
    10. Hoogenraad CC
    11. Akhmanova A

    (2008) STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER

    Current Biology 18:177–182.

    https://doi.org/10.1016/j.cub.2007.12.050

    • PubMed
    • Google Scholar
18. 1. Hetz C
    2. Chevet E
    3. Oakes SA

    (2015) Proteostasis control by the unfolded protein response

    Nature Cell Biology 17:829–838.

    https://doi.org/10.1038/ncb3184

    • PubMed
    • Google Scholar
19. 1. Hirosumi J
    2. Tuncman G
    3. Chang L
    4. Görgün CZ
    5. Uysal KT
    6. Maeda K
    7. Karin M
    8. Hotamisligil GS

    (2002) A central role for JNK in obesity and insulin resistance

    Nature 420:333–336.

    https://doi.org/10.1038/nature01137

    • PubMed
    • Google Scholar
20. 1. Hotamisligil GS

    (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease

    Cell 140:900–917.

    https://doi.org/10.1016/j.cell.2010.02.034

    • PubMed
    • Google Scholar
21. 1. Hotamisligil GS

    (2017) Inflammation, metaflammation and immunometabolic disorders

    Nature 542:177–185.

    https://doi.org/10.1038/nature21363

    • PubMed
    • Google Scholar
22. 1. Hu P
    2. Shimoji S
    3. Hart GW

    (2010) Site-specific interplay between O-GlcNAcylation and phosphorylation in cellular regulation

    FEBS Letters 584:2526–2538.

    https://doi.org/10.1016/j.febslet.2010.04.044

    • PubMed
    • Google Scholar
23. 1. Kammoun HL
    2. Chabanon H
    3. Hainault I
    4. Luquet S
    5. Magnan C
    6. Koike T
    7. Ferré P
    8. Foufelle F

    (2009) GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice

    Journal of Clinical Investigation 119:1201–1215.

    https://doi.org/10.1172/JCI37007

    • PubMed
    • Google Scholar
24. 1. Kang S
    2. Dahl R
    3. Hsieh W
    4. Shin A
    5. Zsebo KM
    6. Buettner C
    7. Hajjar RJ
    8. Lebeche D

    (2016) Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders

    Journal of Biological Chemistry 291:5185–5198.

    https://doi.org/10.1074/jbc.M115.705012

    • PubMed
    • Google Scholar
25. 1. Kars M
    2. Yang L
    3. Gregor MF
    4. Mohammed BS
    5. Pietka TA
    6. Finck BN
    7. Patterson BW
    8. Horton JD
    9. Mittendorfer B
    10. Hotamisligil GS
    11. Klein S

    (2010) Tauroursodeoxycholic Acid may improve liver and muscle but not adipose tissue insulin sensitivity in obese men and women

    Diabetes 59:1899–1905.

    https://doi.org/10.2337/db10-0308

    • PubMed
    • Google Scholar
26. 1. Liou J
    2. Kim ML
    3. Heo WD
    4. Jones JT
    5. Myers JW
    6. Ferrell JE
    7. Meyer T

    (2005) STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

    Current Biology 15:1235–1241.

    https://doi.org/10.1016/j.cub.2005.05.055

    • PubMed
    • Google Scholar
27. 1. Liou J
    2. Fivaz M
    3. Inoue T
    4. Meyer T

    (2007) Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion

    Proceedings of the National Academy of Sciences 104:9301–9306.

    https://doi.org/10.1073/pnas.0702866104

    • PubMed
    • Google Scholar
28. 1. Lopez JJ
    2. Albarran L
    3. Gómez LJ
    4. Smani T
    5. Salido GM
    6. Rosado JA

    (2016) Molecular modulators of store-operated calcium entry

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1863:2037–2043.

    https://doi.org/10.1016/j.bbamcr.2016.04.024

    • Google Scholar
29. 1. Lynes EM
    2. Simmen T

    (2011) Urban planning of the endoplasmic reticulum (ER): How diverse mechanisms segregate the many functions of the ER

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1813:1893–1905.

    https://doi.org/10.1016/j.bbamcr.2011.06.011

    • Google Scholar
30. 1. López E
    2. Salido GM
    3. Rosado JA
    4. Berna-Erro A

    (2012) Unraveling STIM2 function

    Journal of Physiology and Biochemistry 68:619–633.

    https://doi.org/10.1007/s13105-012-0163-1

    • PubMed
    • Google Scholar
31. 1. Maus M
    2. Cuk M
    3. Patel B
    4. Lian J
    5. Ouimet M
    6. Kaufmann U
    7. Yang J
    8. Horvath R
    9. Hornig-Do HT
    10. Chrzanowska-Lightowlers ZM
    11. Moore KJ
    12. Cuervo AM
    13. Feske S

    (2017) Store-Operated Ca2+ Entry Controls Induction of Lipolysis and the Transcriptional Reprogramming to Lipid Metabolism

    Cell Metabolism 25:698–712.

    https://doi.org/10.1016/j.cmet.2016.12.021

    • PubMed
    • Google Scholar
32. 1. Meikle PJ
    2. Summers SA

    (2017) Sphingolipids and phospholipids in insulin resistance and related metabolic disorders

    Nature Reviews Endocrinology 13:79–91.

    https://doi.org/10.1038/nrendo.2016.169

    • PubMed
    • Google Scholar
33. 1. Nakatani Y
    2. Kaneto H
    3. Kawamori D
    4. Yoshiuchi K
    5. Hatazaki M
    6. Matsuoka TA
    7. Ozawa K
    8. Ogawa S
    9. Hori M
    10. Yamasaki Y
    11. Matsuhisa M

    (2005) Involvement of endoplasmic reticulum stress in insulin resistance and diabetes

    Journal of Biological Chemistry 280:847–851.

    https://doi.org/10.1074/jbc.M411860200

    • PubMed
    • Google Scholar
34. 1. Oh-Hora M
    2. Yamashita M
    3. Hogan PG
    4. Sharma S
    5. Lamperti E
    6. Chung W
    7. Prakriya M
    8. Feske S
    9. Rao A

    (2008) Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance

    Nature Immunology 9:432–443.

    https://doi.org/10.1038/ni1574

    • PubMed
    • Google Scholar
35. 1. Orci L
    2. Ravazzola M
    3. Le Coadic M
    4. Shen WW
    5. Demaurex N
    6. Cosson P

    (2009) From the Cover: STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum

    Proceedings of the National Academy of Sciences 106:19358–19362.

    https://doi.org/10.1073/pnas.0911280106

    • PubMed
    • Google Scholar
36. 1. Ozawa K
    2. Miyazaki M
    3. Matsuhisa M
    4. Takano K
    5. Nakatani Y
    6. Hatazaki M
    7. Tamatani T
    8. Yamagata K
    9. Miyagawa J
    10. Kitao Y
    11. Hori O
    12. Yamasaki Y
    13. Ogawa S

    (2005) The endoplasmic reticulum chaperone improves insulin resistance in type 2 diabetes

    Diabetes 54:657–663.

    https://doi.org/10.2337/diabetes.54.3.657

    • PubMed
    • Google Scholar
37. 1. Ozcan U
    2. Cao Q
    3. Yilmaz E
    4. Lee AH
    5. Iwakoshi NN
    6. Ozdelen E
    7. Tuncman G
    8. Görgün C
    9. Glimcher LH
    10. Hotamisligil GS

    (2004) Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes

    Science 306:457–461.

    https://doi.org/10.1126/science.1103160

    • PubMed
    • Google Scholar
38. 1. Ozcan U
    2. Yilmaz E
    3. Ozcan L
    4. Furuhashi M
    5. Vaillancourt E
    6. Smith RO
    7. Görgün CZ
    8. Hotamisligil GS

    (2006) Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes

    Science 313:1137–1140.

    https://doi.org/10.1126/science.1128294

    • PubMed
    • Google Scholar
39. 1. Ozcan L
    2. Wong CC
    3. Li G
    4. Xu T
    5. Pajvani U
    6. Park SK
    7. Wronska A
    8. Chen BX
    9. Marks AR
    10. Fukamizu A
    11. Backs J
    12. Singer HA
    13. Yates JR
    14. Accili D
    15. Tabas I

    (2012) Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity

    Cell Metabolism 15:739–751.

    https://doi.org/10.1016/j.cmet.2012.03.002

    • PubMed
    • Google Scholar
40. 1. Ozcan L
    2. Cristina de Souza J
    3. Harari AA
    4. Backs J
    5. Olson EN
    6. Tabas I

    (2013) Activation of calcium/calmodulin-dependent protein kinase II in obesity mediates suppression of hepatic insulin signaling

    Cell Metabolism 18:803–815.

    https://doi.org/10.1016/j.cmet.2013.10.011

    • PubMed
    • Google Scholar
41. 1. Ozcan L
    2. Tabas I

    (2016) Calcium signalling and ER stress in insulin resistance and atherosclerosis

    Journal of Internal Medicine 280:457–464.

    https://doi.org/10.1111/joim.12562

    • PubMed
    • Google Scholar
42. 1. Phillips MJ
    2. Voeltz GK

    (2016) Structure and function of ER membrane contact sites with other organelles

    Nature Reviews Molecular Cell Biology 17:69–82.

    https://doi.org/10.1038/nrm.2015.8

    • PubMed
    • Google Scholar
43. 1. Poenie M
    2. Tsien R

    (1986)

    Fura-2: a powerful new tool for measuring and imaging [Ca2+]i in single cells

    Progress in Clinical and Biological Research 210:53–56.

    • PubMed
    • Google Scholar
44. 1. Pozo-Guisado E
    2. Casas-Rua V
    3. Tomas-Martin P
    4. Lopez-Guerrero AM
    5. Alvarez-Barrientos A
    6. Martin-Romero FJ

    (2013) Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1

    Journal of Cell Science 126:3170–3180.

    https://doi.org/10.1242/jcs.125054

    • PubMed
    • Google Scholar
45. 1. Prakriya M
    2. Lewis RS

    (2015) Store-Operated Calcium Channels

    Physiological Reviews 95:1383–1436.

    https://doi.org/10.1152/physrev.00020.2014

    • PubMed
    • Google Scholar
46. 1. Rong X
    2. Albert CJ
    3. Hong C
    4. Duerr MA
    5. Chamberlain BT
    6. Tarling EJ
    7. Ito A
    8. Gao J
    9. Wang B
    10. Edwards PA
    11. Jung ME
    12. Ford DA
    13. Tontonoz P

    (2013) LXRs regulate ER stress and inflammation through dynamic modulation of membrane phospholipid composition

    Cell Metabolism 18:685–697.

    https://doi.org/10.1016/j.cmet.2013.10.002

    • PubMed
    • Google Scholar
47. 1. Sharma NK
    2. Das SK
    3. Mondal AK
    4. Hackney OG
    5. Chu WS
    6. Kern PA
    7. Rasouli N
    8. Spencer HJ
    9. Yao-Borengasser A
    10. Elbein SC

    (2008) Endoplasmic reticulum stress markers are associated with obesity in nondiabetic subjects

    The Journal of Clinical Endocrinology & Metabolism 93:4532–4541.

    https://doi.org/10.1210/jc.2008-1001

    • PubMed
    • Google Scholar
48. 1. ADIPOGen Consortium
    2. CARDIOGRAMplusC4D Consortium
    3. CKDGen Consortium
    4. GEFOS Consortium
    5. GENIE Consortium
    6. GLGC
    7. ICBP
    8. International Endogene Consortium
    9. LifeLines Cohort Study
    10. MAGIC Investigators
    11. MuTHER Consortium
    12. PAGE Consortium
    13. ReproGen Consortium
    14. Shungin D
    15. Winkler TW
    16. Croteau-Chonka DC
    17. Ferreira T
    18. Locke AE
    19. Mägi R
    20. Strawbridge RJ
    21. Pers TH
    22. Fischer K
    23. Justice AE
    24. Workalemahu T
    25. Wu JMW
    26. Buchkovich ML
    27. Heard-Costa NL
    28. Roman TS
    29. Drong AW
    30. Song C
    31. Gustafsson S
    32. Day FR
    33. Esko T
    34. Fall T
    35. Kutalik Z
    36. Luan J
    37. Randall JC
    38. Scherag A
    39. Vedantam S
    40. Wood AR
    41. Chen J
    42. Fehrmann R
    43. Karjalainen J
    44. Kahali B
    45. Liu CT
    46. Schmidt EM
    47. Absher D
    48. Amin N
    49. Anderson D
    50. Beekman M
    51. Bragg-Gresham JL
    52. Buyske S
    53. Demirkan A
    54. Ehret GB
    55. Feitosa MF
    56. Goel A
    57. Jackson AU
    58. Johnson T
    59. Kleber ME
    60. Kristiansson K
    61. Mangino M
    62. Leach IM
    63. Medina-Gomez C
    64. Palmer CD
    65. Pasko D
    66. Pechlivanis S
    67. Peters MJ
    68. Prokopenko I
    69. Stančáková A
    70. Sung YJ
    71. Tanaka T
    72. Teumer A
    73. Van Vliet-Ostaptchouk JV
    74. Yengo L
    75. Zhang W
    76. Albrecht E
    77. Ärnlöv J
    78. Arscott GM
    79. Bandinelli S
    80. Barrett A
    81. Bellis C
    82. Bennett AJ
    83. Berne C
    84. Blüher M
    85. Böhringer S
    86. Bonnet F
    87. Böttcher Y
    88. Bruinenberg M
    89. Carba DB
    90. Caspersen IH
    91. Clarke R
    92. Daw EW
    93. Deelen J
    94. Deelman E
    95. Delgado G
    96. Doney AS
    97. Eklund N
    98. Erdos MR
    99. Estrada K
    100. Eury E
    101. Friedrich N
    102. Garcia ME
    103. Giedraitis V
    104. Gigante B
    105. Go AS
    106. Golay A
    107. Grallert H
    108. Grammer TB
    109. Gräßler J
    110. Grewal J
    111. Groves CJ
    112. Haller T
    113. Hallmans G
    114. Hartman CA
    115. Hassinen M
    116. Hayward C
    117. Heikkilä K
    118. Herzig KH
    119. Helmer Q
    120. Hillege HL
    121. Holmen O
    122. Hunt SC
    123. Isaacs A
    124. Ittermann T
    125. James AL
    126. Johansson I
    127. Juliusdottir T
    128. Kalafati IP
    129. Kinnunen L
    130. Koenig W
    131. Kooner IK
    132. Kratzer W
    133. Lamina C
    134. Leander K
    135. Lee NR
    136. Lichtner P
    137. Lind L
    138. Lindström J
    139. Lobbens S
    140. Lorentzon M
    141. Mach F
    142. Magnusson PK
    143. Mahajan A
    144. McArdle WL
    145. Menni C
    146. Merger S
    147. Mihailov E
    148. Milani L
    149. Mills R
    150. Moayyeri A
    151. Monda KL
    152. Mooijaart SP
    153. Mühleisen TW
    154. Mulas A
    155. Müller G
    156. Müller-Nurasyid M
    157. Nagaraja R
    158. Nalls MA
    159. Narisu N
    160. Glorioso N
    161. Nolte IM
    162. Olden M
    163. Rayner NW
    164. Renstrom F
    165. Ried JS
    166. Robertson NR
    167. Rose LM
    168. Sanna S
    169. Scharnagl H
    170. Scholtens S
    171. Sennblad B
    172. Seufferlein T
    173. Sitlani CM
    174. Smith AV
    175. Stirrups K
    176. Stringham HM
    177. Sundström J
    178. Swertz MA
    179. Swift AJ
    180. Syvänen AC
    181. Tayo BO
    182. Thorand B
    183. Thorleifsson G
    184. Tomaschitz A
    185. Troffa C
    186. van Oort FV
    187. Verweij N
    188. Vonk JM
    189. Waite LL
    190. Wennauer R
    191. Wilsgaard T
    192. Wojczynski MK
    193. Wong A
    194. Zhang Q
    195. Zhao JH
    196. Brennan EP
    197. Choi M
    198. Eriksson P
    199. Folkersen L
    200. Franco-Cereceda A
    201. Gharavi AG
    202. Hedman ÅK
    203. Hivert MF
    204. Huang J
    205. Kanoni S
    206. Karpe F
    207. Keildson S
    208. Kiryluk K
    209. Liang L
    210. Lifton RP
    211. Ma B
    212. McKnight AJ
    213. McPherson R
    214. Metspalu A
    215. Min JL
    216. Moffatt MF
    217. Montgomery GW
    218. Murabito JM
    219. Nicholson G
    220. Nyholt DR
    221. Olsson C
    222. Perry JR
    223. Reinmaa E
    224. Salem RM
    225. Sandholm N
    226. Schadt EE
    227. Scott RA
    228. Stolk L
    229. Vallejo EE
    230. Westra HJ
    231. Zondervan KT
    232. Amouyel P
    233. Arveiler D
    234. Bakker SJ
    235. Beilby J
    236. Bergman RN
    237. Blangero J
    238. Brown MJ
    239. Burnier M
    240. Campbell H
    241. Chakravarti A
    242. Chines PS
    243. Claudi-Boehm S
    244. Collins FS
    245. Crawford DC
    246. Danesh J
    247. de Faire U
    248. de Geus EJ
    249. Dörr M
    250. Erbel R
    251. Eriksson JG
    252. Farrall M
    253. Ferrannini E
    254. Ferrières J
    255. Forouhi NG
    256. Forrester T
    257. Franco OH
    258. Gansevoort RT
    259. Gieger C
    260. Gudnason V
    261. Haiman CA
    262. Harris TB
    263. Hattersley AT
    264. Heliövaara M
    265. Hicks AA
    266. Hingorani AD
    267. Hoffmann W
    268. Hofman A
    269. Homuth G
    270. Humphries SE
    271. Hyppönen E
    272. Illig T
    273. Jarvelin MR
    274. Johansen B
    275. Jousilahti P
    276. Jula AM
    277. Kaprio J
    278. Kee F
    279. Keinanen-Kiukaanniemi SM
    280. Kooner JS
    281. Kooperberg C
    282. Kovacs P
    283. Kraja AT
    284. Kumari M
    285. Kuulasmaa K
    286. Kuusisto J
    287. Lakka TA
    288. Langenberg C
    289. Le Marchand L
    290. Lehtimäki T
    291. Lyssenko V
    292. Männistö S
    293. Marette A
    294. Matise TC
    295. McKenzie CA
    296. McKnight B
    297. Musk AW
    298. Möhlenkamp S
    299. Morris AD
    300. Nelis M
    301. Ohlsson C
    302. Oldehinkel AJ
    303. Ong KK
    304. Palmer LJ
    305. Penninx BW
    306. Peters A
    307. Pramstaller PP
    308. Raitakari OT
    309. Rankinen T
    310. Rao DC
    311. Rice TK
    312. Ridker PM
    313. Ritchie MD
    314. Rudan I
    315. Salomaa V
    316. Samani NJ
    317. Saramies J
    318. Sarzynski MA
    319. Schwarz PE
    320. Shuldiner AR
    321. Staessen JA
    322. Steinthorsdottir V
    323. Stolk RP
    324. Strauch K
    325. Tönjes A
    326. Tremblay A
    327. Tremoli E
    328. Vohl MC
    329. Völker U
    330. Vollenweider P
    331. Wilson JF
    332. Witteman JC
    333. Adair LS
    334. Bochud M
    335. Boehm BO
    336. Bornstein SR
    337. Bouchard C
    338. Cauchi S
    339. Caulfield MJ
    340. Chambers JC
    341. Chasman DI
    342. Cooper RS
    343. Dedoussis G
    344. Ferrucci L
    345. Froguel P
    346. Grabe HJ
    347. Hamsten A
    348. Hui J
    349. Hveem K
    350. Jöckel KH
    351. Kivimaki M
    352. Kuh D
    353. Laakso M
    354. Liu Y
    355. März W
    356. Munroe PB
    357. Njølstad I
    358. Oostra BA
    359. Palmer CN
    360. Pedersen NL
    361. Perola M
    362. Pérusse L
    363. Peters U
    364. Power C
    365. Quertermous T
    366. Rauramaa R
    367. Rivadeneira F
    368. Saaristo TE
    369. Saleheen D
    370. Sinisalo J
    371. Slagboom PE
    372. Snieder H
    373. Spector TD
    374. Stefansson K
    375. Stumvoll M
    376. Tuomilehto J
    377. Uitterlinden AG
    378. Uusitupa M
    379. van der Harst P
    380. Veronesi G
    381. Walker M
    382. Wareham NJ
    383. Watkins H
    384. Wichmann HE
    385. Abecasis GR
    386. Assimes TL
    387. Berndt SI
    388. Boehnke M
    389. Borecki IB
    390. Deloukas P
    391. Franke L
    392. Frayling TM
    393. Groop LC
    394. Hunter DJ
    395. Kaplan RC
    396. O'Connell JR
    397. Qi L
    398. Schlessinger D
    399. Strachan DP
    400. Thorsteinsdottir U
    401. van Duijn CM
    402. Willer CJ
    403. Visscher PM
    404. Yang J
    405. Hirschhorn JN
    406. Zillikens MC
    407. McCarthy MI
    408. Speliotes EK
    409. North KE
    410. Fox CS
    411. Barroso I
    412. Franks PW
    413. Ingelsson E
    414. Heid IM
    415. Loos RJ
    416. Cupples LA
    417. Morris AP
    418. Lindgren CM
    419. Mohlke KL

    (2015) New genetic loci link adipose and insulin biology to body fat distribution

    Nature 518:187–196.

    https://doi.org/10.1038/nature14132

    • PubMed
    • Google Scholar
49. 1. Soboloff J
    2. Rothberg BS
    3. Madesh M
    4. Gill DL

    (2012) STIM proteins: dynamic calcium signal transducers

    Nature Reviews Molecular Cell Biology 13:549–565.

    https://doi.org/10.1038/nrm3414

    • PubMed
    • Google Scholar
50. 1. Vaeth M
    2. Maus M
    3. Klein-Hessling S
    4. Freinkman E
    5. Yang J
    6. Eckstein M
    7. Cameron S
    8. Turvey SE
    9. Serfling E
    10. Berberich-Siebelt F
    11. Possemato R
    12. Feske S

    (2017) Store-Operated Ca2+ Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming

    Immunity 47:664–679.

    https://doi.org/10.1016/j.immuni.2017.09.003

    • PubMed
    • Google Scholar
51. 1. Varadi A
    2. Lebel L
    3. Hashim Y
    4. Mehta Z
    5. Ashcroft SJ
    6. Turner R

    (1999) Sequence variants of the sarco(endo)plasmic reticulum Ca(2+)-transport ATPase 3 gene (SERCA3) in Caucasian type II diabetic patients (UK Prospective Diabetes Study 48)

    Diabetologia 42:1240–1243.

    https://doi.org/10.1007/s001250051298

    • PubMed
    • Google Scholar
52. 1. Vosseller K
    2. Wells L
    3. Lane MD
    4. Hart GW

    (2002) Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes

    PNAS 99:5313–5318.

    https://doi.org/10.1073/pnas.072072399

    • PubMed
    • Google Scholar
53. 1. Wang Y
    2. Li G
    3. Goode J
    4. Paz JC
    5. Ouyang K
    6. Screaton R
    7. Fischer WH
    8. Chen J
    9. Tabas I
    10. Montminy M

    (2012) Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes

    Nature 485:128–132.

    https://doi.org/10.1038/nature10988

    • PubMed
    • Google Scholar
54. 1. Wang M
    2. Kaufman RJ

    (2016) Protein misfolding in the endoplasmic reticulum as a conduit to human disease

    Nature 529:326–335.

    https://doi.org/10.1038/nature17041

    • PubMed
    • Google Scholar
55. 1. Westrate LM
    2. Lee JE
    3. Prinz WA
    4. Voeltz GK

    (2015) Form follows function: the importance of endoplasmic reticulum shape

    Annual Review of Biochemistry 84:791–811.

    https://doi.org/10.1146/annurev-biochem-072711-163501

    • PubMed
    • Google Scholar
56. 1. Williams RT
    2. Manji SS
    3. Parker NJ
    4. Hancock MS
    5. Van Stekelenburg L
    6. Eid JP
    7. Senior PV
    8. Kazenwadel JS
    9. Shandala T
    10. Saint R
    11. Smith PJ
    12. Dziadek MA

    (2001) Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins

    Biochemical Journal 357:673–685.

    https://doi.org/10.1042/bj3570673

    • PubMed
    • Google Scholar
57. 1. Wilson CH
    2. Ali ES
    3. Scrimgeour N
    4. Martin AM
    5. Hua J
    6. Tallis GA
    7. Rychkov GY
    8. Barritt GJ

    (2015) Steatosis inhibits liver cell store-operated Ca²⁺ entry and reduces ER Ca²⁺ through a protein kinase C-dependent mechanism

    The Biochemical Journal 466:379–390.

    https://doi.org/10.1042/BJ20140881

    • PubMed
    • Google Scholar
58. 1. Wu MM
    2. Luik RM
    3. Lewis RS

    (2007) Some assembly required: constructing the elementary units of store-operated Ca2+ entry

    Cell Calcium 42:163–172.

    https://doi.org/10.1016/j.ceca.2007.03.003

    • PubMed
    • Google Scholar
59. 1. Xiao C
    2. Giacca A
    3. Lewis GF

    (2011) Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and beta-cell dysfunction in humans

    Diabetes 60:918–924.

    https://doi.org/10.2337/db10-1433

    • PubMed
    • Google Scholar
60. 1. Yang X
    2. Ongusaha PP
    3. Miles PD
    4. Havstad JC
    5. Zhang F
    6. So WV
    7. Kudlow JE
    8. Michell RH
    9. Olefsky JM
    10. Field SJ
    11. Evans RM

    (2008) Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance

    Nature 451:964–969.

    https://doi.org/10.1038/nature06668

    • PubMed
    • Google Scholar
61. 1. Yang X
    2. Qian K

    (2017) Protein O-GlcNAcylation: emerging mechanisms and functions

    Nature Reviews Molecular Cell Biology 18:452–465.

    https://doi.org/10.1038/nrm.2017.22

    • PubMed
    • Google Scholar
62. 1. Zhu-Mauldin X
    2. Marsh SA
    3. Zou L
    4. Marchase RB
    5. Chatham JC

    (2012) Modification of STIM1 by O-linked N-acetylglucosamine (O-GlcNAc) attenuates store-operated calcium entry in neonatal cardiomyocytes

    The Journal of Biological Chemistry 287:39094–39106.

    https://doi.org/10.1074/jbc.M112.383778

    • PubMed
    • Google Scholar

Author details

1. Ana Paula Arruda

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Contributed equally with

   Benedicte Mengel Pers

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6179-2687

2. Benedicte Mengel Pers

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Present address

   The Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Ana Paula Arruda

   Competing interests

   No competing interests declared

3. Günes Parlakgul

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Ekin Güney

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Ted Goh

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

6. Erika Cagampan

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

7. Grace Yankun Lee

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

8. Renata L Goncalves

   Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

9. Gökhan S Hotamisligil

   1. Department of Genetics and Complex Diseases, Sabri Ülker Center, Harvard TH Chan School of Public Health, Boston, United States
   2. Broad Institute of MIT and Harvard, Cambridge, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   ghotamis@hsph.harvard.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2906-1897

• 3,528

  views

• 652

  downloads

• 43

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.