Obesity is the result of a mismatch between energy intake and energy expenditure. Although a sedentary lifestyle can contribute to obesity development, the consumption of sugar-sweetened beverages and high amounts of saturated fat in foods has been linked to the risk to become obese and develop metabolic disorders (Hall et al., 2012). Over the last decades, the intake of sugar-based beverages has clearly increased worldwide, and from recent surveys it has become clear that consumption of both sugar-sweetened beverages and saturated fats (especially from snack foods) exceeds recommended daily levels (Popkin et al., 2012). It is, therefore, of utmost importance to understand how fat and sugar affect energy balance.

The hypothalamus is an important brain area in regulating energy balance (Elmquist and Flier, 2004; Schwartz and Porte, 2005). Under normal conditions the hypothalamus senses whole body energy demands via nutrient, neuronal and hormonal signaling and adjusts feeding behavior and energy expenditure via the production of orexigenic and anorexigenic peptides. For example leptin, the hormone secreted by adipose tissue in proportion to fat mass, activates the release of α-melanocyte stimulating hormone [α-MSH; derived from pro-opiomelanocortin (POMC)] and inhibits the release of the orexigenic neuropeptide Y (NPY) and agouti related peptide (AgRP) (Sahu, 2011). During obesity, however, the function of intracellular organelles in AGRP/NPY or POMC neurons like the endoplasmic reticulum (ER) was shown to be impaired leading to disturbed leptin signaling and energy imbalance (Hosoi et al., 2008; Zhang et al., 2008; Ozcan et al., 2009; Cakir et al., 2013; Ramírez and Claret, 2015).

The ER is a complex membrane network responsible for the synthesis and folding of various transmembrane and secreted proteins (Westrate et al., 2015). Accumulation of toxic, misfolded proteins in the ER leads to ER stress and activation of the unfolded protein response (UPR). The UPR is a network of pathways controlled by three sensors; PKR-like ER kinase (PERK), the activating transcription factor 6 (ATF6) and the inositol-requiring protein-1 alpha (IREα). Activation of these pathways induces the expression of genes that lead to the expansion of the ER, reduction of protein translation and increase of protein folding capacity promoting cell survival or induces apoptosis (Walter and Ron, 2011; Lee and Ozcan, 2014). Upon PERK activation, activating transcription factor 4 (ATF4) mRNA is translated which increases the transcription of specific UPR target genes, including C/EBP homologous protein (CHOP) (Harding et al., 2000; Han et al., 2013). Immunoglobulin-heavy-chain-binding protein (BiP) mRNA is increased upon ATF6 activation and splicing of unspliced X box binding protein 1 to spliced XBP1 (sXBP1/usXBP1) occurs upon IREα activation (Yoshida et al., 2001). Both usXBP1 and sXBP1 were related to cell viability under ER stress conditions. Under severe ER stress, IREα activation can also lead to degradation of mRNAs and miRNAs and cell apoptosis via the c-Jun N-terminal kinase (JNK) pathway (Todd et al., 2008). DP5 (Death protein 5/harakiri) and FasL (Fas ligand) are genes expressed downstream of JNK indicating activation of the IREα—apoptotic pathway (Schenkel, 2004; Guan et al., 2006; Ma et al., 2007).

Long term high fat diet (HFD) feeding, resulting in profound body weight gain, induces hypothalamic ER stress characterized by increased protein levels of UPR markers like p-PERK, p-IREα, and phospho-eukaryotic initiation factor 2 alpha (p-eIF2α) (Ozcan et al., 2009; Cakir et al., 2013). Activation of UPR pathways was also reported when fatty acids (FA) [arachidic acid, palmitic acid, or ceramide] were directly supplied to the brain via intracerebroventricular (ICV) infusion in rodents (Milanski et al., 2009; Contreras et al., 2014) or when administered to murine neuronal cell lines (Choi et al., 2010) implying a more direct role for FA in hypothalamic ER stress induction. Although many of the HFD used also contain considerable amounts of sugar, the effects of sugar alone on the ER stress induction has not been studied so far.

The fact that long term HFD feeding, but also ICV infusions of FA, induce hypothalamic ER stress marker expression points to the idea that nutrients like FA can induce hypothalamic ER stress. However, it is not clear whether other nutrients or the increased adiposity reflecting a positive energy status or a combination of nutrients and energy status affects hypothalamic ER stress. We therefore determined whether overfeeding/fasting status and its interaction with diet-induced obesity affected genetic markers for ER stress in the hypothalamus. We exposed male Wistar rats to a free choice high-fat high-sugar (fcHFHS) diet or chow for 1 week followed by overnight ad libitum feeding, fat/sugar deprivation or fasting. In addition, we investigated if fat and sugar have a direct effect on the ER stress markers in the hypothalamus by intracarotic infusions of Intralipids (IL), IL and glucose, or glucose to the brain of overnight fasted lean rats. For both experiments, mRNA of different UPR markers was measured using RT-PCR as an indication for the induction of hypothalamic ER stress.

We showed that short term exposure to a fcHFHS diet, followed by mild food restriction overnight induces hypothalamic ER stress in rats as characterized by an increase in sXPB1/usXBP1 mRNA ratio in hypothalamus of fcHFHS fed rats compared to chow fed rats. Moreover, we showed that lipids, directly infused towards the brain of overnight fasted rats, are able to induce a similar response. As non-restricted ad libitum fcHFHS-diet fed or totally fasted rats do not show altered ratios, these data point to an interaction of lipid exposure to the brain and a negative energy balance in ER stress induction. In addition, we observed an increase in ATF4 mRNA when animals were ad libitum fed the fcHFHS diet which is abolished when rats are either provided with 10 g of chow overnight or totally fasted. In addition, high fat consumers on the fcHFHS diet have higher ATF4 mRNA, pointing to a direct role for fat intake in the increase in hypothalamic ATF4 mRNA. Indeed, ATF4 mRNA is also increased when animals are directly infused with lipids or lipids and glucose towards the brain, but not when glucose is infused alone.

An increase in sXPB1/usXBP1mRNA ratio implies activation of the IREα pathway (Yoshida et al., 2001; Calfon et al., 2002). The activation of IREα pathway due to HFD-feeding has been reported earlier. However, this was after long-term (8 or 20 weeks) exposure (Ozcan et al., 2009; Won et al., 2009). Here we showed for the first time that short term exposure to a fcHFHS diet is enough to induce activation of this pathway, but only under mild food restriction.

It is unclear why animals on a fcHFHS diet display this response only when food restricted while absent when animals are ad libitum fed overnight. One possibility might be that ER stress was induced via anabolic processes as illustrated by the observation that overnight fasting and subsequent refeeding increases sXBP1 mRNA and protein in the liver (Deng et al., 2013) and in the hypothalamus of mice (Williams et al., 2014). It is also possible that protective mechanisms play a role under ad libitum conditions. For example, we observed recently that 1 week fcHFHS diet increases beta- oxidation genes in the hypothalamus (Rijnsburger et al., 2016), and since enhanced beta-oxidation has been reported to counter palmitate-induced ER stress in in vitro models (McFadden et al., 2014), it is possible that we do not observe increased splicing of XPB1 under ad libitum feeding because of counter-active regulatory fatty acid oxidation. In line, we observed no changes in fatty acid oxidation genes after lipid infusion directly to the brain while it does induce XBP1 splicing (M. Rijnsburger, unpublished data). Further research is needed to determine the exact role of energy status and nutrient sensing on hypothalamic ER stress induction.

We observed a clear increase in hypothalamic ATF4 mRNA in rats on the fcHFHS diet while being ad libitum fed or when infused with intralipids via the carotic artery compared to vehicle infusions. However, we did not observe induction of downstream targets. It might be possible that increased ATF4 mRNA expression in our models is associated with other general stress related events within a cell i.e., amino-acid or glucose deprivation (Siu et al., 2002) or less protein availability (which has been shown in muscle to result in UPR activation; Deldicque et al., 2010). An additional effect of the fcHFHS diet is reduced chow—and thus protein—intake compared to chow controls, it is well possible that this might be the cause for the changes in ATF4 mRNA expression observed in our study. It is unclear at this point whether this increase in ATF4 mRNA will result in increased ATF4 protein. Interestingly, overexpression and inhibition of ATF4 in the hypothalamus induces hepatic insulin resistance and improves hepatic insulin sensitivity, respectively (Zhang et al., 2013). In addition, a recent study showed that ATF4 deletion in AGRP neurons of the hypothalamus specifically protects against high fat diet induced weight gain and insulin resistance (Deng et al., 2016). Together, this suggests an important role for hypothalamic ATF4 in the regulation of energy metabolism. Interestingly, we previously reported hepatic insulin resistance in rats on a fcHFHS diet (Diepenbroek et al., 2017), which could well be related to the observed increase in hypothalamic ATF4 mRNA.

Under severe ER stress, IREα activation can also lead to cell apoptosis via the JNK pathway (Todd et al., 2008) and to activation of apoptotic genes (Urano et al., 2000; Guan et al., 2006; Kim et al., 2006; Ma et al., 2007). We therefore measured DP5 and FasL mRNA, target genes of JNK pathway (Schenkel, 2004; Guan et al., 2006; Ma et al., 2007). However, 1 week fcHFHS diet did not result in activation of apoptotic gene expression. It could well be that this is due to the duration of the diet, as exposure to the diet for a longer period induces apoptosis accompanied by obesity and other metabolic disturbances (Ozcan et al., 2009; Won et al., 2009).

Interestingly a decrease in DP5 mRNA expression was observed in the fcHFHS group after overnight mild food restriction (10 g chow intake). That might be related to the increase in sXBP1 mRNA under the same conditions. Since both are regulated by IREα activation, the observed alterations suggest a shift from cell death related pathways to the induction of cell survival mechanisms. Like DP5, FasL mRNA can be also induced upon IREα/JNK activation during neuronal apoptosis (Le-Niculescu et al., 1999; Schenkel, 2004; Chen et al., 2015). However, no change was observed in FasL expression indicating that FasL does not play a major role in our experimental setting. In addition, a glucose infusion directly to the brain lowered FasL mRNA expression when compared to a vehicle control infusion, however what this means physiologically remains to be determined.

In summary, a fcHFHS diet and overnight fat and sugar availability affects the mRNA expression of hypothalamic ER stress related UPR markers. Overall, the UPR markers seemed to be a sensitive sensor of fatty acid availability as well as nutrient load. More studies are necessary to define the exact role of nutrients in induction of UPR intermediates that play a role in cellular metabolism and viability.

EB, AB, and Sl designed experiments. EB, MR, LE, UU, and Sl performed experiments. EB, AB, and Sl prepared the manuscript. WS, MR, LE, and UU edited the manuscript. The entire study was supervised by AB and Sl.