Ghrelin is a 28 amino acid acylated enteroendocrine peptide produced mainly in the stomach, which has been isolated and named based on its ability to stimulate growth hormone secretion (1). One of the main properties of ghrelin, independent from growth hormone secretagogue activity, is stimulation of feeding and body weight gain (2–4). Importantly, acylation of the serine 3 residue in the ghrelin's N-terminal by octanoic acid is necessary for binding of the growth hormone secretagogue receptor 1 (GHSR1), designated as ghrelin receptor (5) and biological activity including stimulation of food intake (6–8). However, the active, acylated form of ghrelin is unstable in the circulation and is degraded to desacyl-ghrelin (9, 10). The half-life pharmacokinetics properties of acyl-ghrelin in human plasma are about 10 min (11). The preservation of the acylated ghrelin form in the circulation appears, hence, as a key factor for maintaining its role in regulation of appetite and energy balance. Several mechanisms are involved in the regulation of ghrelin signaling during its production and receptor activation, which can be exploited for development of ghrelin-based therapy of altered appetite (12). Recently, a new regulatory factor of the ghrelin signaling has been identified, which is ghrelin-reactive immunoglobulins (Ig) (13). This review is the first attempt to summarize the data on ghrelin-reactive IgG obtained since their initial identification and the following studies in animal models of altered appetite and energy balance.

### METHODS

The review focuses mainly on the data from five papers, all reporting affinity kinetics of ghrelin- and desacyl-ghrelin-reactive IgG. They include a study of patients with hyperphagic obesity and anorexia nervosa (AN) and of *ob/ob* mice by Takagi et al. (13); two studies by François et al. in high-fat diet (HFD)-induced obese mice (14) and in rats with methotrexate (MTX)-induced anorexia (15), a study by Lucas et al. in obese Zucker rats (16) as well as a Legrand et al. study in mice after chronic food-restriction and activity-based anorexia (17). In all mentioned studies, the same protocols of IgG plasma extraction and affinity kinetic assay by the surface plasmon resonance were used as was previously described in details (18). All measurements of ghrelin and desacyl-ghrelin peptides in above mentioned studies were performed using corresponding kits from Mitsubishi Chemical Med Corp. (Tokyo, Japan), selectively detecting acyl- and desacyl-ghrelin with resulting plasma ratios of 1:3 to 1:6 in humans. In the text, we use the terms of ghrelin and acyl-ghrelin as synonyms. For the statistical analysis of ghrelin and ghrelin-reactive IgG properties between different animal models and patients, they are shown as means of folds of changes vs. corresponding controls. In this analysis, we combined two animal models of anorexia including ABA mice and MTX rats and three animal models of obesity including *ob/ ob* mice, HFD-induced obese mice, and obese Zucker rats. Group differences shown in **Tables 1**–**4** and **Figure 1** were analyzed using statistical tests indicated in the legends. We also used the Pearson's test to analyze correlations between plasma levels of

*Human patients: OB, obese; AN, anorexia nervosa. Rodent models of obesity: ob/ ob mice; HFD, high fat diet-induced obese mice and Zucker rats. Rodent models of anorexia and starvation: ABA, activity-based anorexia in mice; FTR, feeding-time restriction in mice; and MTX, methotrexate-induced anorexia in rats. Ctrl, healthy human or rodent controls in corresponding row. Student's t-test or Mann–Whitney test, \*p* < *0.05; \*\*p* < *0.01; # p* < *0.10. ANOVA with Tukey's posttest or Kruskal–Wallis with Dunn's posttest \$ p* < *0.05, \$\$p* < *0.01, \$\$\$p* < *0.001.*

ghrelin or desacyl-ghrelin and plasma levels and affinity (KD values) of corresponding IgG.

### DETECTION OF GHRELIN-REACTIVE IgG

Ghrelin-reactive IgG were first shown to be naturally present in plasma of healthy humans and rodents in 2008 by Fetissov et al. (19). Further studies confirmed and extended this initial finding by including female patients with restrictive AN (20) and with hyperphagic obesity (13). Moreover, a study using plasma samples of a large number of healthy male (*n* = 562) and female (*n* = 636) adolescents also revealed an ubiquitous presence of ghrelin-reactive autoantibodies of both IgG and IgM classes, which mean plasma levels were slightly elevated in girls vs. boys (21). In rodents, ghrelin-reactive IgG were detected in both rats (15, 19) and mice (13, 14, 17). Since the rodents studies were performed only in males, presence of possible sex differences of ghrelin-reactive IgG was not explored. However, it is likely that similarly to humans they may also be increased in female rodents who naturally display elevated plasma levels of total IgG and IgM as well as increased levels of autoantibodies reactive with other neuropeptides, e.g., α-melanocyte-stimulating hormone (α-MSH) (22).

Ghrelin-reactive IgG were also measured in the hypothalamic and liver tissue of C57Bl6 mice, showing their levels at the limit of detection (13). However, if levels of ghrelin-reactive IgG in these tissues of lean mice were stratified according to their exposure to a restraint stress in an EchoMRI instrument, which they had undergone prior to tissue sample for the analysis of their body composition, an increase is observed in both tissues and a decrease in plasma of stressed animals. It suggests that ghrelin-reactive IgG may help transportation of ghrelin mobilized during stress from plasma to its tissue targets.

Desacyl-ghrelin-reactive IgG are also present in humans (13, 20) and rodents (15, 17). Although some part of desacyl-ghrelinreactive IgG may bind to acyl-ghrelin, they should also have distinct paratopes, because levels of desacyl-ghrelin IgG are often higher than of acyl-ghrelin IgG in human plasma (13, 20) and also because they have different affinity kinetics properties (13). The exact epitopes responsible for plasma IgG binding to ghrelin and desacyl-ghrelin remain to be been studied.

Both "free" and "total" ghrelin- and desacyl-ghrelin-reactive IgG are detected in plasma of humans and rodents. While "free" IgG are measured in physiological buffer, the "total" levels are measured in a high salt buffer, which dissociate immune complexes typically resulting in increased "total" vs. "free" levels of IgG. Accordingly, increased levels of free/total ratios may reflect an increase in IgG available to bind ghrelin or desacyl-ghrelin (**Table 2**).

### ORIGIN AND REGULATION OF GHRELIN-REACTIVE IgG

Ghrelin-reactive IgG are natural autoantibodies and, therefore, are produced by germ-like B-cells as a part of native immunity (23). As was postulated by Avrameas, natural autoantibodies may participate in a complex regulatory network, not necessarily

#### Table 2 | Plasma levels of IgG reactive with acyl-ghrelin and desacyl-ghrelin in humans and rodents.

*Human patients: OB, obese; AN, anorexia nervosa. Rodent models of obesity: ob/ob mice; HFD, high fat diet-induced obese mice and Zucker rats. Rodent models of anorexia and starvation: ABA, activity-based anorexia in mice; FTR, feeding-time restriction in mice; and MTX, methotrexate-induced anorexia in rats. Ctrl, healthy human or rodent controls in corresponding row. Student's t-test or Mann–Whitney test, \*p* < *0.05; \*\*p* < *0.01; \*\*\*p* < *0.001; # p* < *0.10. ANOVA with Tukey's posttest or Kruskal–Wallis test with Dunns' posttest, \$ p* < *0.05; ABA vs. FTR £ p* < *0.05.*

#### Table 3 | Affinity kinetics of acyl-ghrelin-reactive IgG in humans and rodents.

*Human patients: OB, obese; AN, anorexia nervosa. Rodent models of obesity: ob/ob mice; HFD, high fat diet-induced obese mice and Zucker rats. Rodent models of anorexia and starvation: ABA, activity-based anorexia in mice; FTR, feeding-time restriction in mice; and MTX, methotrexate-induced anorexia in rats. Ctrl, healthy human or rodent controls in corresponding row. ANOVA with Tukey's posttest or Kruskal–Wallis test with Dunn's posttest, \$ p* < *0.05; AN vs. OB §§p* < *0.01. Student's t-test or Mann–Whitney test vs. Ctrl \*p* < *0.05; \*\*p* < *0.01, vs. RFA £ p* < *0.05.*

directly related to the immune function but contributing to the homeostatic control (24). The production and affinity maturation of IgG are influenced by a variety of antigens and non-specific immune-stimulatory or inhibitory factors such as cytokines and steroid hormones. Moreover, increased plasma levels of ghrelin-reactive IgG in rats can be induced acutely by gastric electrical stimulation (25), suggesting that they can be stored

and released upon a physiological stimulus. It is of interest that gastric electrical stimulation activated c-fos production in numerous lymphoid cells of the gastric mucosa surrounding the enteroendocrine ghrelin-synthetizing cells (26). Strong inhibition of ghrelin-reactive IgG levels in plasma is observed in rats after treatment with MTX, an immunosuppressive agent, which induces intestinal mucositis (15).

Table 4 | Affinity kinetics of desacyl-ghrelin-reactive IgG in humans and rodents.

*Human patients: OB, obese; AN, anorexia nervosa. Rodent models of obesity: ob/ob mice; HFD, high fat diet-induced obese mice and Zucker rats. Rodent models of anorexia and starvation: ABA, activity-based anorexia in mice; FTR, feeding-time restriction in mice; and MTX, methotrexate-induced anorexia in rats. Ctrl, healthy human or rodent controls in corresponding row. ANOVA with Tukey's posttest or Kruskal–Wallis test with Dunn's posttest, \$ p* < *0.05; \$\$p* < *0.01. Student's t-test or Mann–Whitney test, vs. Ctrl \*p* < *0.05; # p* < *0.10.*

A particular role in production of IgG and other classes of Ig can be played by antigens derived from gut microbiota as a constitutive part of the gastro-intestinal tract in all animals (27). The gut bacterial antigens may also be involved in regulation of production and properties of ghrelin-reactive IgG. In fact, germ-free rats showed elevated plasma levels of ghrelin-reactive IgG (19), supporting a role of some intestinal antigens in tolerization toward ghrelin (28). The molecular basis linking microbial antigens with peptides may relate to the concept of molecular mimicry by cross-reactive autoantibody production (29). Indeed, sequence homology between ghrelin and several proteins from commensal gut microorganisms was shown, e.g., of seven consecutive amino acids present in a protein from *Enterococcus faecalis* (19). A role of molecular mimicry was recently validated as a mechanism underlying production of α-MSH-reactive IgG induced by caseinolytic peptidase B (ClpB) homolog protein from *Escherichia coli* (30). ClpB contains a discontinuous six amino acid homology with α-MSH; immunization of mice with ClpB generates α-MSH-cross reactive IgG (30). Plasma presence of the IgA class of ghrelin-reactive IgG also points to their origin triggered by luminal antigens (19).

Beside the stimulation of antigens from commensal gut microbiota, pathogenic and environmental microorganisms may possibly influence production of ghrelin-reactive IgG. In fact, plasma levels of ghrelin-reactive IgG in healthy adolescents correlate strongly with both IgG and IgM directed against influenza A virus (21). Although the initial *in silico* search for five consecutive amino acids sequence homology did not reveal matches between ghrelin and the Influenza viruses (19), a discontinuous eight amino acid homology for ghrelin is present in the PB1-F2 influenza A protein.

Another example of ghrelin-reactive IgG comes from studies of children with idiopathic short stature by the Lewinski group.

Figure 1 | Relative to control (Ctrl) values (1.0) changes in plasma ghrelin and desacyl ghrelin as well as affinity of their reactive IgG in patients and animal models of obesity and anorexia. Changes in plasma levels of acyl-ghrelin (A) and desacyl-ghrelin (C). Changes in affinity (dissociation equilibrium constant, KD) of plasmatic IgG for acyl-ghrelin (B) and desacyl ghrelin (D). AN, anorexia nervosa patients; An, animal models of anorexia; OB, obese patients or animals. Mann–Whitney tests \*\*\**p* < 0.001, \*\**p* < 0.01, and \**p* < 0.05.

They showed that prevalence of high level IgG reactive with several peptide hormones, including ghrelin, orexin A, leptin, and α-MSH combined, was associated with an increased incidence of *Helicobacter pylori* and/or *Candida albicans* (31). A follow-up study showed that mean plasma levels of ghrelin-reactive IgG were also elevated in children with short stature, but they were not significantly different in patients with growth hormone deficiency (32). Since *H. pylori* is only residing in the stomach, which is the main source of ghrelin, it is of interest to explore possible role of *H. pylori* in production of ghrelin-reactive IgG. In fact, cure of *H. pylori* in humans was accompanied by increased plasma levels of ghrelin (33).

### FUNCTIONAL ROLE OF GHRELIN-REACTIVE IgG

An established physiological role of IgG is to protect against infection including their ability to directly neutralize antigens and to trigger cell lysis *via* activation of the complement. While presence of IgG reactive with some peptide hormones have been detected in plasma of healthy humans (34), their possible functional role remained unknown, as previously reviewed (35). Ghrelin-reactive IgG were the first IgG revealing that they have a functional role in the peptide signaling by protecting ghrelin from degradation in plasma (13). In fact, as was shown by an *in vitro* assay, depletion of plasma from IgG resulted in an almost complete loss of acyl-ghrelin, while their reintroduction allowed its full recovery (13). Thus, the main functional role of ghrelinreactive IgG appears to protect ghrelin from degradation.

Such protection did not reduce the orexigenic activity of ghrelin, because administrations of ghrelin together with plasmatic IgG, stimulated food intake and even enhanced it when IgG were derived from plasma of obese humans or mice (13). Such enhancing effect was explained by a slight, about three times, increase of IgG affinity for ghrelin and the evidence that such IgG are able to transport more active ghrelin in obese patients. Moreover, the KD values of ghrelin-reactive IgG remained in the micromolar range, which would preclude their competition with the nanomolar affinity of ghrelin receptor binding (36).

We show here that KD values of IgG for ghrelin correlate positively with plasma ghrelin concentrations in obese and AN patients as well as in *ob/ob* mice (**Figures 2A,B**). These correlations demonstrate that a decrease in IgG affinity for ghrelin (increase in KD) corresponds to higher ghrelin levels and suggest that properties of ghrelin-reactive IgG may at least partly underlie individual differences in plasma ghrelin levels. An inverse relation is unlikely, because increased antigen concentration should stimulate the affinity maturation of IgG. We also find that plasma levels of ghrelin-reactive free IgG as well as ratios of free/total IgG levels correlate positively with plasma ghrelin (**Figures 2C,D**). In contrast, ghrelin-reactive total IgG correlate negatively with plasma ghrelin (**Figure 2E**). Taken together, these correlations support a carrier role of plasmatic IgG for ghrelin.

Figure 2 | Examples of correlations between affinity and levels of ghrelin-reactive IgG and plasma concentrations of acyl-ghrelin. Correlations between the values of dissociation equilibrium constants (KD) of plasmatic IgG reactive with ghrelin and acyl ghrelin in obese and anorectic patients and controls (A) and in *ob/ob* and lean mice (B). Correlations between plasma levels of ghrelin-reactive free IgG (C) as well as ratios of their free/total levels (D) in ob/ob and lean mice. Correlations between plasma levels of ghrelin-reactive total IgG in Zucker rats (E). *R*-squared and *p*-values for Pearson's correlation tests are shown, \*\**p* < 0.01 and \**p* < 0.05.

Considering increased functional effects of ghrelin in immune complex with IgG vs. ghrelin alone, the accompanying changes in plasma ghrelin levels during altered energy metabolism can be a "mirror" reflection of the ghrelin's functional activity. Accordingly, increased plasma levels of ghrelin, e.g., in AN patients and in animal models of anorexia (**Figure 1**), may signify ghrelin's inability to form stable immune complexes with IgG and, hence, may result in a functional deficiency of the ghrelin signaling or "ghrelin resistance." Such explanation may appear at first glance as paradoxical, but it gains further support from the corroborating data revealing an enhancing role of IgG in signaling by other peptide hormones such as α-MSH (37, 38).

Thus, the changes in IgG affinity kinetics resulting in a slight increase of affinity can be considered as a gain of function in ghrelin signaling. Taking in account important functional consequences of affinity changes in ghrelin-reactive IgG, it is clear that a simple measurement of their plasma levels is not sufficient and should be combined with the affinity kinetics analysis. Nevertheless, affinity changes should still be taken with caution as a putative biomarker, as long as we do not know if they involve or not any epitope changes that may prevent the availability of the ghrelin N-terminal, necessary for the GHSR1 binding. We do not yet know if presence of IgG in immune complex with ghrelin may play an allosteric role in ghrelin receptor activation, a phenomenon suggested for IgG reactive with α-MSH (37, 38).

To summarize these data, presence of IgG at normal levels and with physiological micromolar affinity for ghrelin in humans appears as a homeostatic factor in regulation of ghrelin signaling. Accordingly, any changes in these factors should lead to alteration of the ghrelin signaling with resulting effects on appetite and body weight regulation. Therefore, in order to facilitate the data interpretation toward an enhanced or diminished ghrelin signaling, the following algorithm can be proposed.

For the enhanced ghrelin signaling one of several changes can be observed:

By the opposite, for the diminished ghrelin signaling, the following changes can be present:

While the functional role of ghrelin-reactive IgG with regard to ghrelin's orexigenic effect can be interpreted as facilitating, the corresponding role of IgG reactive with desacyl-ghrelin is uncertain. As discussed above, IgG may bind to the common central and the C-terminal parts of ghrelin and desacyl-ghrelin and, hence, may transport both types of peptides. In fact, absorption studies of ghrelin- and desacyl-ghrelin-reactive IgG confirmed that they can bind both peptides (20).

Although some studies showed potential anorexigenic effects of desacyl-ghrelin (39, 40), its functional role in the regulation of appetite remains uncertain including direct functional consequences of desacyl-ghrelin transport by IgG toward an unknown putative receptor (41). One alternative possibility is that desacyl-ghrelin may compete with acyl-ghrelin for protective IgG and, thereby, desacyl-ghrelin may have an anorexigenic effect by diminishing the ghrelin signaling *via* reduced formation of ghrelin/IgG immune complexes. Such possibility is of functional importance because starvation preferentially upregulates desacylghrelin, e.g., its ratios to acyl-ghrelin can be increased from 1:20 in *ad libitum* fed mice to more than 1:70 in starved mice (**Table 1**) (17). Functional significance of the affinity kinetics changes in desacyl-ghrelin-reactive IgG is also difficult to interpret owning the same problem of the uncertain role of the desacyl-ghrelin peptide. Whether such changes are different from those obtained for acyl-ghrelin, it suggests that they may concern antibodies primarily targeting the desacylated N-terminal of ghrelin. It is, nevertheless, possible that decreased affinity of IgG for desacylghrelin may be favorable for desacyl-ghrelin competition with ghrelin for ghrelin-protective IgG and, hence, may result in reduced orexigenic activity of ghrelin. Furthermore, we have seen that KD values of desacyl-ghrelin IgG correlate positively with plasma levels of desacyl-ghrelin in animals with anorexia. Significant correlations were also found between plasma levels of desacyl-ghrelin reactive IgG and desacyl-ghrelin in patients with obesity and AN. Such correlations suggest that, similar to acyl-ghrelin, desacyl-ghrelin plasma concentrations can also be regulated by desacyl-ghrelin-reactive IgG.

#### GHRELIN-REACTIVE-IgG IN OBESITY

Obesity is typically accompanied by increased food intake, suggesting a possible role of ghrelin in the mechanism of hyperphagia (42). Indeed, obese subjects are more sensitive than lean subjects to increase their food intake after ghrelin administration (43). A particular situation may exist in non-hyperphagic HFD-induced obesity in mice that are less sensitive to the ghrelin's orexigenic effect (44). In obese subjects, basal plasma levels of ghrelin measured as total ghrelin (acyl-ghrelin + desacyl-ghrelin) were found low (45, 46) or normal when measured selectively for acyl-ghrelin (13, 47, 48) (**Table 1**). Thus, increased food intake in common obesity cannot be related to increased ghrelin concentrations. There are, nevertheless, some exceptions showing elevated plasma ghrelin in hyperphagic obese patients with the Prader–Willi syndrome (PWS) (49) and hyperphagic obese Zucker rats (50, 51). However, our study showed that obese Zucker rats display elevated desacyl-ghrelin, while acyl-ghrelin remains normal (16) (**Table 1**).

IgG in obese patients showed their ability to better protect ghrelin from degradation by an *in vitro* test and to increase its orexigenic activity after intraperitoneal administrations in rats (13). Such ghrelin's enhancing properties of IgG in obesity can be explained by its increased affinity, as discussed above. Similarly, increased affinity of IgG for ghrelin was found in genetic animal models of obesity including both *ob/ob* mice (13) and Zucker rats (16) (**Table 3**). Obesity in both rodent's models is due to the deficient leptin signaling and is characterized by hyperphagia. In mice that developed obesity after 2 months of HFD, IgG was also characterized by increased affinity for ghrelin (**Table 3**); however, these mice are not hyperphagic. In contrast to other animal models of obesity and to obese patients, HFD-obese mice also showed an increase in affinity of desacyl-ghrelin IgG (**Table 4**), but the significance of such change is not clear. The affinity kinetic properties of the association and dissociation rates leading to increased affinity were all different between humans and animal models of obesity including increased small *Ka* in obese patients, a decrease of small *Kd* in *ob/ob* mice and Zucker rats, and no significant changes in HFD obese mice (**Table 3**).

Although plasma levels of ghrelin-reactive IgG in obese patients and in all animal models reviewed here were slightly lower than in their non-obese controls (**Table 2**), the free/total ratios of ghrelin-reactive IgG were increased in patients and in Zucker rats (**Table 2**). Interestingly, a recent conference report revealed elevated plasma levels of ghrelin-reactive IgG in children with PWS (52). If confirmed, it will be the only so far known pathological conditions characterized by increased production of ghrelin-reactive IgG.

Taken together, these data suggest that obesity development leads to increased carrying properties of IgG for ghrelin in both obese humans and in animal models of obesity *via* increased affinity (**Figure 1**). Such changes in ghrelin-reactive IgG properties may represent a mechanistic factor underlying enhanced ghrelin signaling leading to hyperphagia and increased adiposity.

### GHRELIN-REACTIVE-IgG IN ANOREXIA

Anorexia can be a primary problem in patient with eating disorders such as in restrictive AN, or it can be symptomatic during a chronic disease worsening its outcome. Ghrelin production and its plasma concentration are typically elevated during chronic starvation, accompanied or not by anorexia in both humans and

orexigenic effects. Desacyl-ghrelin may lower orexigenic effect *via* competing with ghrelin for ghrelin-reactive IgG resulting in increased ghrelin degradation. Moreover, decreased affinity of IgG for desacyl ghrelin may favor its dissociation from immune complexes and completion with ghrelin. The origin of changes in affinity of ghrelin- and desacyl ghrelin-reactive IgG is currently unknown but may potentially depend on antigenic stimulation from dysbiotic gut microbiota associated

with long-term nutritional modifications in anorexia and obesity.

experimental animals (13, 17, 53–56). Such elevation of plasma ghrelin suggests its homeostatic role in the long-term regulation of energy balance aimed at increased food intake, once food will be available (42). This homeostatic control is obviously not working in anorectic humans and rodents, who display functional "ghrelin resistance." A role of plasmatic IgG in mechanisms of "ghrelin resistance" during anorexia can be suggested based on changes in their properties in an opposite way to obesity. In fact, patients with AN display lower affinity than in controls (**Table 3**), and many AN patients display also low plasma levels of ghrelin-reactive IgG (20). In the ABA and MTX rodent models of anorexia, we did not observe low affinity of IgG for ghrelin but it was present for desacyl-ghrelin (**Tables 3** and **4**). Such decrease of affinity was detected only in ABA mice but not in FTR mice that were starved but did not develop spontaneous anorexia, suggesting a possible contribution of desacyl-ghrelin-reactive IgG in the anorectic phenotype. MTX-treated rats, which develop severe anorexia, were also characterized by strong inhibition of ghrelinreactive IgG levels (15). As discussed above, lower levels and affinity of IgG for both ghrelin and desacyl-ghrelin may decrease ghrelin's orexigenic effects. Such possibility was not, however, experimentally validated.

Whether IgG may improve ghrelin's orexigenic effects was tested in the ABA model in mice, but the orexigenic effects of treatments were limited to the 3 h of daily feeding time, which was not sufficient to fully evaluate their enhancing roles (17). In any case, only IgG from obese mice were efficient to significantly improve ghrelin's orexigenic effect.

These data suggest that factors which specifically or nonspecifically decrease plasma levels and affinity of ghrelin-reactive IgG may diminish ghrelin's orexigenic effects and, hence, will contribute to the mechanisms of anorexia *via* functional "ghrelin resistance."

#### CONCLUSION

The main conclusions that can be drawn after reviewing these data are the existence of a functional link between ghrelin-reactive IgG and plasma ghrelin levels as well as a physiological role of ghrelin-reactive IgG in modulating ghrelin's biological activities including conditions of altered energy balance. A summary

#### REFERENCES

**Figure 3** schematically illustrates the postulated link between ghrelin-reactive IgG and regulation of appetite whereas changes in IgG properties during obesity and anorexia may enhance or diminish ghrelin signaling, while desacyl-ghrelin may compete with ghrelin for IgG which protect ghrelin from deacylation.

Data analysis from patients and several animal models of altered energy balance shows that both obese humans and animals display lower levels of ghrelin-reactive IgG characterized by increased affinity. In contrast, patients with AN and anorectic rodents all show lower affinity of ghrelin- and desacyl-ghrelinreactive IgG, respectively, the changes which were not observed in non-anorectic chronically starved mice. Such changes in properties of both ghrelin- and desacyl-ghrelin-reactive IgG in patients with obesity and anorexia suggest that they are not a direct consequence of the positive or negative energy balance *per se* but may be a result of some typical changes associated with altered regulation of energy metabolism impacting on the autoantibody production. Although the mechanisms of such common changes are currently unknown, they may potentially involve modifications of gut microbiota leading to altered production of ghrelin-like antigenic molecules. Indeed, typical, but not identical response of gut microbiota to starvation or an obesogenic diet may potentially explain similar autoantibody response including production of ghrelin-reactive IgG, which may contribute to the long-term regulation of host energy metabolisms by gut microbiota (57). Further studies are needed to identify potential ghrelin-mimetic antigens in gut microbiota and to analyze the effects of altered energy balance on their production.

### AUTHOR CONTRIBUTIONS

SF wrote the manuscript, NL prepared the figures and tables, NL and RL analyzed the data and contributed to manuscript preparation.

#### FUNDING

The reviewed studies were partly supported from the Region of Haute Normandie, France, Fonds Français Alimentation et Santé, Fondation Charles Nicolle and Nutriset, France.

conditioned medium. *Endocrinology* (2010) 151(10):4765–75. doi:10.1210/ en.2010-0412

**Conflict of Interest Statement:** SF is a co-founder of TargEDys SA and NL and RL are currently its employees.

*Copyright © 2017 Fetissov, Lucas and Legrand. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Combination of Selective Immunoassays and Mass Spectrometry to Characterize Preproghrelin-Derived Peptides in Mouse Tissues

Rim Hassouna1, 2 †, Dominique Grouselle1 †, Giovanni Chiappetta<sup>3</sup> , Joanna Lipecka<sup>1</sup> , Oriane Fiquet <sup>1</sup> , Catherine Tomasetto<sup>4</sup> , Joëlle Vinh<sup>3</sup> , Jacques Epelbaum1, 5 and Virginie Tolle<sup>1</sup> \*

<sup>1</sup> Centre de Psychiatrie et Neurosciences, UMR-S 894 Institut National de la Santé et de la Recherche Médicale, Université Paris Descartes, Sorbonne Paris Cité, Paris, France, <sup>2</sup> Department of Pediatrics, Naomi Berrie Diabetes Center, Columbia University Medical Center, New York, NY, USA, <sup>3</sup> ESPCI Paris, PSL Research University, Spectrométrie de Masse Biologique et Protéomique (SMPB), CNRS USR 3149, Paris, France, <sup>4</sup> UMR-7104 Centre Nationnal de la Recherche Scientifique/U596, Institut National de la Santé et de la Recherche Médicale, Institut de génétique et de biologie moléculaire et cellulaire, Université de Strasbourg, Illkirch, France, <sup>5</sup> UMR 7179 Centre Nationnal de la Recherche Scientifique, MNHN, Adaptive Mechanism and Evolution (MECADEV), Brunoy, France

#### Edited by:

Hubert Vaudry, University of Rouen, France

#### Reviewed by:

Sergueï O. Fetissov, University of Rouen, France Roy G. Smith, Scripps Research Institute, USA

#### \*Correspondence:

Virginie Tolle<EMAIL_ADDRESS>These authors have contributed

†

#### Specialty section:

equally to this work.

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 30 January 2017 Accepted: 29 March 2017 Published: 20 April 2017

#### Citation:

Hassouna R, Grouselle D, Chiappetta G, Lipecka J, Fiquet O, Tomasetto C, Vinh J, Epelbaum J and Tolle V (2017) Combination of Selective Immunoassays and Mass Spectrometry to Characterize Preproghrelin-Derived Peptides in Mouse Tissues. Front. Neurosci. 11:211. doi: 10.3389/fnins.2017.00211 Preproghrelin is a prohormone producing several preproghrelin-derived peptides with structural and functional heterogeneity: acyl ghrelin (AG), desacyl ghrelin (DAG), and obestatin. The absence of selective and reliable assays to measure these peptides simultaneously in biological samples has been a limitation to assess their real proportions in tissues and plasma in physiological and pathological conditions. We aimed at reliably measure the ratio between the different preproghrelin-derived peptides in murine tissues using selective immunoassays combined with a highly sensitive mass spectrometry method. AG-, DAG-, and obestatin-immunopositive fractions from the gastrointestinal tract of adult wild-type and ghrelin-deficient mice were processed for analysis by mass spectrometry (MS) with a Triple Quadrupole mass spectrometer. We found that DAG was predominant in mouse plasma, however it only represented 50% of total ghrelin (AG+DAG) production in the stomach and duodenum. Obestatin plasma levels accounted for about 30% of all circulating preproghrelin-derived peptides, however, it represented <1% of total preproghrelin-derived peptides production (AG+DAG+Obestatin) in the stomach. Assays were validated in ghrelin-deficient mice since neither ghrelin nor obestatin immunoreactivities were detected in their stomach, duodenum nor plasma. MS analyses confirmed that obestatin-immunoreactivity in stomach corresponded to the C-terminal amidated form of the peptide but not to des(1–10)-obestatin, nor to obestatin-Gly. In conclusion, specificity of ghrelin and obestatin immunoreactivities in gastrointestinal tissues using selective immunoassays was validated by MS. Obestatin was less abundant than AG or DAG in these tissues. Whether this is due to inefficient processing rate of preproghrelin into mature obestatin in gastrointestinal mouse tissues remains elusive.

Keywords: acyl ghrelin, desacyl ghrelin, obestatin, immunoreactivity, mass spectrometry

## INTRODUCTION

Preproghrelin is a complex prohormone that, upon posttranslational processing, leads to the production of several derived peptides with structural and functional heterogeneity. Ghrelin is a 28 amino acid peptide originating from the stomach (Kojima et al., 1999; Hosoda et al., 2000; Tomasetto et al., 2000) and identified as the endogenous ligand of the Growth Hormone Secretagogue Receptor (GHS-R; Howard et al., 1996). The addition of an acyl-group by the Ghrelin-O-Acyl-Transferase (GOAT; Yang et al., 2008), enables ghrelin (Acyl-ghrelin, AG) to stimulate GH secretion and appetite (Tolle et al., 2001, 2002). Another endogenous form of ghrelin is desacyl ghrelin (DAG) reported as the most abundant form in plasma (Hosoda et al., 2000). Its specific roles are to regulate glucose, lipid, and bone metabolism (Delhanty et al., 2013). Obestatin is a 23 amino acid amidated peptide derived from the same precursor as ghrelin. Originally isolated as the endogenous ligand for the GPR39 and described as an anorexigenic factor in rodents (McKee et al., 1997; Zhang et al., 2005), its physiological relevance has since been questioned (Zhang et al., 2005; Bresciani et al., 2006; Lauwers et al., 2006; Seoane et al., 2006; Yamamoto et al., 2007). Both DAG and obestatin interact pharmacologically with AG to modulate food intake, GH secretion or glucose metabolism through yet unidentified receptors (Hassouna et al., 2012; Delhanty et al., 2013; Gargantini et al., 2013). Previous studies found equimolar ratios of plasma AG and obestatin levels in the rat (Zhang et al., 2005; Zizzari et al., 2007), consistent with both peptides being processed from the same prohormone. However, evidence that tissue specific splicing variant encoding obestatin but not ghrelin exist in humans suggests that obestatin could also be produced independently of ghrelin (Seim et al., 2009).

With regard to the literature, many issues remain to be addressed concerning obestatin: its main source of production in the body and its abundance relative to ghrelin as well as its molecular form and way of processing. Obestatin was initially extracted from rat stomach and found in rat plasma (Zhang et al., 2005). In addition, obestatin immunoreactivity was detected in a number of human tissues using immunohistochemistry (Grönberg et al., 2008) and in cultured pancreatic islets in vitro (Granata et al., 2008). However, other studies failed to detect significant amounts of obestatin in rat plasma or stomach using radioimmunoassay (RIA) coupled to High Performance Liquid Chromatography (HPLC; Bang et al., 2007; Mondal et al., 2008).

The absence of selective and reliable assays to measure all three preproghrelin-derived peptides (AG, DAG, and obestatin) simultaneously in biological samples is an obstacle to further characterization of their specific physiological and pathophysiological functions. In this study, we developed selective immunoassays combined to a highly sensitive targeted mass spectrometry method in order to reliably measure and characterize the ratios of the different preproghrelin-derived peptides in mice. Preproghrelin gene deficient mice that do not produce AG or DAG (Hassouna et al., 2014) were used as negative controls to further validate the immunoreactivity and mass spectrometry assays.

## MATERIALS AND METHODS

#### Animals

Dissections were performed on 7–12 weeks old preproghrelin deficient (ghrl−/−) mice and wild type (ghrl+/+) littermates backcrossed on the C57BL/6J genetic background as previously reported (Hassouna et al., 2014). Mice were housed in a room under controlled illumination (0700–1900 h) and temperature (22–24◦C) and had free access to food and water. Offsprings were genotyped by PCR amplification of tail DNA. All experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and were approved by the Animal Experimentation Committee of Paris Descartes University (agreement number 03422.02).

#### Peptides

Peptides used as standards in liquid chromatography (LC) were provided by NeoMPS (Strasbourg, France): rat/mouse acyl ghrelin (AG) and desacyl ghrelin (DAG), rat/mouse amidated obestatin (obestatin-NH2), Des(1–10)-obestatin and obestatin-Gly (Sequences presented in Table S1 and Figure S1).

### Dissection, Extraction, and Purification of Tissue Samples

Gastric epithelia, 1 cm proximal duodenum, small intestine and colon were collected and the tissues were extracted in 2N acetic acid during 10 min at 90◦C, sonicated and frozen at −80◦C for 24 h. The homogenate was centrifuged 20 min at 12,000 g at 4◦C. Supernatants were lyophilized and further dissolved in phosphate assay buffer. Extracts were first filtered on a 10 kDa filter (Amicon Ultra, Millipore, Molsheim, France) then purified using SepPak C18 columns (Waters, Saint-Quentin-en-Yvelines, France). Briefly, the supernatants were loaded onto a SepPak C18 cartridge pre-equilibrated in 0.1% trifluoroacetic acid (TFA). The samples were desalted with aqueous TFA 0.1% and eluted with an acetonitrile gradient (10–100%).

### Plasma Collection and Processing

Blood samples were collected from trunk blood on EDTA (1 mg/ml) and PHMB (0.4 mM final), a serine protease inhibitor and centrifuged at 1,000 g during 10 min at 4◦C. Plasma were immediately acidified with HCl (0.1 M final) and stored at −80◦C.

### Hormone Assays

Immunoreactivities were measured in plasma, whole tissue and SepPak fractions (10–70 and 100%) with selective sandwich immunoassays for AG, DAG (SPIbio Bertin pharma, A05118 for the acyl form and A05117 for the desacyl form, Montignyle-Bretonneux, France) and competitive assay for obestatin (Phoenix Pharmaceuticals, Burlingame, USA). Immunoreactive fractions were further analyzed by Mass Spectrometry (MS).

### Mass Spectrometry

Mass Spectrometry (MS) analyses were performed in the Selected Reaction Monitoring (SRM) mode using a high pressure nanoLC (U3000 RSLC, Thermo Fisher Scientific) coupled to a triple quadrupole (QqQ) mass spectrometer (TSQ VantageTM, Thermo Fisher Scientific, San Jose, CA, USA) in nano-ESI mode. Briefly, peptides were loaded and desalted on a C18 cartridge (C18 PepMap, 3 mm, 100 A◦ , 75 mm i.d., 2 cm length) using a loading buffer containing 0.05% aq TFA/acetonitrile 98:2 (v/v) at 10 µL/min. Peptides were then separated on a C18 analytical column (C18 PepMap, 2 mm, 100 A◦ , 75 mm i.d., 15 cm length) with a 60 min gradient from 99% A [0.1% aq formic acid/acetonitrile 98:2 (v/v)] to 50% B [0.1% aq formic acid/acetonitrile 10:90 (v/v)] at 300 nL/min. Standard was injected before each series of experiments. Blank runs were interposed until necessary to avoid peptide carry-over effects. QqQ parameters were set as follows: first and third quadrupole widths set at 0.7, scan time 200 ms/transition and total dwell time 3 s (method performed in unscheduled mode). The transitions for AG (retention time 29.1 min) were 553.1 (precursor MH6<sup>+</sup> 6 ) → 513.3 (y4); 641.4 (y5); 712.4;(y6); 809.5 (y7) 906.5(y8). The transitions for DAG (retention time 20.0 min) were 532.1 (precursor MH6<sup>+</sup> 6 ) → 513.3 (y4); 641.4 (y5); 712.4; (y6); 809.5 (y7); 906.5(y8). The transitions for obestatin-NH<sup>2</sup> (retention time 38.3 min) were 630.8 (precursor MH5<sup>+</sup> 5 ) → 262.1(b2); 416.2 (y4); 553.3 (y5); 681.4;(y6); 972.5 (y8). The transitions for obestatin-Gly (retention time 33.4 min) were 858.8 (precursor MH3<sup>+</sup> 3 ) → 262.1 (b2); 333.2 (b3); 473.2 (y5); 610.3 (y6) 1029.5(y9). The transitions for des(1–10)-obestatin (retention time 17.9 min) were 476.9 (precursor MH3<sup>+</sup> 3 ) → 201.1 (b2); 553.2 (y5); 681.3;(y6); 809.5 (y7); 972.5(y8) (Supplementary Table S1).

#### RESULTS

### Preproghrelin-Derived Peptides Immunoreactivities in the Gastrointestinal Tract and Plasma

In adult mice, stomach is known to be the major source of ghrelin, however the main source of obestatin remains elusive. Accordingly, since ghrelin gene is widely expressed throughout the gastro-intestinal (GI) tract, we thus measured all preproghrelin-derived peptides in the different portions of the GI tract (stomach, duodenum, intestine, and colon) in mice using selective antibodies (Figure S1). Amongst all preproghrelin-derived peptides, AG and DAG were the most abundant forms found in stomach. In this tissue, AG represented 50%, DAG 50%, and obestatin <1% of all preproghrelinderived peptides. Although their proportions varied in the different sections of the GI tract, ghrelin (AG and DAG) remained predominant compared to obestatin. In duodenum, AG represented 34%, DAG 38%, and obestatin 29% of all preproghrelin-derived peptides. Differences in proportions of obestatin relative to ghrelin may partly be due to the utilization of different techniques to detect specific immunoreactivities and revelation procedures (sandwich enzymoimmunoassays for ghrelin vs. competitive radioimmunoassay for obestatin). Whereas, AG and DAG levels were gradually decreasing from caudal to distal region of the GI tract, obestatin concentrations were slightly higher in the duodenum than in the stomach (**Table 1**). Both the intestine and colon produced small amounts of all three peptides (data not shown).

Previous studies using competition assays indicated that DAG accounts for 80–90% of total circulating ghrelin (Hosoda et al., 2000). In the present study, DAG accounted for nearly 80% of total circulating ghrelin (i.e., AG+DAG) and 60% of total circulating preproghrelin-derived peptides (i.e., AG+DAG+Obestatin; **Table 1**). Obestatin plasma levels represented about 30% of all circulating preproghrelin-derived peptides, which is much higher than its proportion in gastrointestinal tissues (**Table 1**).

### Mass Spectrometry (MS) Analysis of Preproghrelin-Derived Peptides in ghrl+/+ and ghrl−/− mice

Selected Reaction Monitoring (SRM) method was set up using synthetic peptides AG, DAG, Obestatin-NH2, Des(1–10)- Obestatin and Obestatin-Gly. The SRM method selected the best five transitions obtained by testing all the theoretic "y" and "b" ion fragments from MH3<sup>+</sup> 3 to MH6<sup>+</sup> 6 precursors (**Figure 1**).

TABLE 1 | Tissue content or concentrations, percentage and molar ratio of preproghrelin-derived peptides immunoreactivities in the stomach, duodenum, and plasma of ghrl+/+ mice (n = 6–10).

AG, Acyl ghrelin; DAG, Desacyl ghrelin; TG, Total Ghrelin (AG+DAG). Limits of detection are 0.03 pmol for AG and DAG and 0.08 pmol for obestatin. Data are expressed as Mean ± SEM, molar ratio or as percentages of total preproghrelin-derived peptides (AG+DAG+obestatin).

The detection limits for AG and DAG was 10–50 fmol and for obestatin-NH<sup>2</sup> and its derivatives 1–5 fmol.

As preproghrelin-derived peptides are highly concentrated in stomach and duodenum, purified SepPak fractions from these tissue protein extracts from both ghrl+/+ and ghrl−/− mice were used for mass spectrometry. Preproghrelin-derived peptides immunoreactivities were retrieved in stomach and duodenum of ghrl+/+ mice but not in the same tissues in ghrl−/− mice (**Table 2** and data not shown). The residual immunoreactivity for obestatin found in two out of six ghrl−/− mice was unspecific, since it did not correspond to the correct specific masses in MS experiments (See below). To decrease the dynamic range and the complexity of the whole protein extract, samples were first depleted to conserve only peptides below 10 kDa. The resulting mixtures were further submitted to solid phase extraction on C18 stationary phase to remove hydrophilic species. According to standard synthetic peptides properties, the endogenous peptides of interest were expected to be eluted using 10–70% acetonitrile. The fractions were submitted to stepwise elution with successive 10% increments of acetonitrile. Immunoreactivity of each preproghrelin-derived peptides (AG, DAG, and Obestatin) was measured in each eluted fraction. In ghrl+/+ mice, 30, 40, and 60% acetonitrile-containing fractions presented the most intense immunoreactivity and were further analyzed by SRM LC-MS. The matched fractions obtained from ghrl−/− mice contained no immunoreactivity for all three preproghrelin-derived peptides and were also analyzed in SRM LC-MS.

Ghrelin and obestatin were detected by mass spectrometry in both ghrl+/+ mice stomach and duodenum, only in 60% acetonitrile fractions (**Table 3**). Thus LC-MS profiles of SRM analyses related to preproghrelin-derived peptides in stomach and duodenum were restricted to 60% acetonitrile fractions from ghrl+/+ and ghrl−/− mice (**Figure 1**). The antibody used in the obestatin immunoassay may detect other forms of obestatin, such as a truncated peptide, des(1–10)-obestatin, and a modified form of obestatin with additional glycine at the C-terminal position, obestatin-Gly. Thus, the presence of des(1–10)-obestatin and obestatin-Gly was also assayed by SRM in order to evaluate a possible antibody cross-reactivity. To develop a specific and

TABLE 2 | Preproghrelin-derived peptides immunoreactivities in the stomach and duodenum of ghrl+/+ and ghrl−/− mice.

Acyl ghrelin, desacyl ghrelin, and obestatin immunoreactivities measured using selective assays are detected in the stomach and duodenum of ghrl+/+ mice (n = 6) but are undetectable in ghrl−/− mice (n = 6). Limits of detection are 10 pg for AG and DAG and 200 pg for obestatin. Data are expressed as Mean ± SEM. UN: Under detection limit. \*Immunoreactivity is unspecific.

sensitive detection method for the peptides of interest, a mix of AG, DAG, obestatin, Des(1–10)-obestatin and obestatin-Gly standards (100 fmol of each) was used to calibrate the SRM method before each analysis. Blanks were run to assure the absence of contaminations from standards before the analysis of samples. Comparison between the chromatographic profiles of SRM analyses related to the fractions eluted at 60% ACN of the stomach and duodenum tissues were compared between ghrl+/+ and ghrl−/− mice (**Figures 1B–F**). As further described below, only native obestatin-NH2 was present in the extracts analyzed (**Table 3**).

The three preproghrelin-derived peptides were present in stomach LC fractions of ghrl+/+ mice. In duodenum LC fractions of ghrl+/+ mice, AG, and obestatin were present while DAG was absent. As shown on **Table 3**, and **Figure 1**, SRM analyses further confirmed that obestatin-immunoreactivity in stomach and duodenum corresponded to the amidated (**Figure 1D**) form of the peptide but not to obestatin-Gly (**Figure 1E**) nor Des(1–10)-obestatin (**Figure 1F**). No MS signal for any preproghrelin-derived peptides was detected in stomach or duodenum of ghrl−/− mice. This further validated the specificity of the spectrometric signal detected (**Figures 1B–F**). In stomach and duodenum extracts from ghrl+/+ and ghrl−/− mice, extracted ion chromatograms related to DAG transitions (**Figure 1C**) presented two peaks instead of one, respectively associated to DAG retention time (21 min) and AG retention time (29 min). The "in source" neutral loss of the acyl group of AG converted partially AG to DAG after LC separation, so that DAG SRM signature can also be detected at AG retention time. On the opposite, when DAG and AG standards are analyzed separately (**Figure 1A**) no signals are detected at DAG retention time with AG transitions.

#### DISCUSSION

Using a combination of selective immunoassays and highly sensitive mass spectrometry, we validate the presence of a specific immunoreactivity signal for amidated obestatin in protein extracts from stomach and duodenum and further characterize the ratio of the different preproghrelin-derived peptides in mouse gastrointestinal tract. Specific presence of each preproghrelin-derived peptide was validated by the lack of signal in preproghrelin deficient mice using both immunodetection and MS.

Until now, very few studies characterized all preproghrelinderived peptides in murine tissues. This can be explained by the lack of sensitive and specific methods to simultaneously detect the three preproghrelin-derived peptides in a given biological sample. Moreover, their relative proportions in tissues and plasma remained unclear. Previous studies using competitive immunoassays reported equimolar ratios of acyl ghrelin and obestatin in rat plasma (Zhang et al., 2005; Zizzari et al., 2007) consistent with a model previewing both peptides obtained by the processing of the same prohormone, while ratios of 2:1–4:1 were reported in humans (Germain et al., 2009, 2010). The reason for such discrepancy is unclear but this could evoke alternative

TABLE 3 | SRM detection of preproghrelin-derived peptides in the different chromatographic fractions (30, 40, and 60% Acetonitrile) in the stomach and duodenum of ghrl+/+ mice.

Acyl ghrelin and obestatin-NH<sup>2</sup> were detected in the stomach and duodenum whereas, desacyl ghrelin was only detected in the stomach LC fractions of ghrl+/+ mice. MS-MS analyses further confirmed that obestatin-immunoreactivity in tissues is specific to the amidated peptide but not to obestatin-Gly nor des(1–10)-obestatin.

splicing and/or different processing mechanism in rodents and humans. Although one study demonstrated that obestatin is produced in gastrointestinal tract in humans (Grönberg et al., 2008), two other studies failed to identify significant amounts of obestatin in rat plasma or stomach by RIA coupled to HPLC (Bang et al., 2007; Mondal et al., 2008) in contrast with the original data from Zhang and collaborators in the rat (Zhang et al., 2005). Moreover, in the study by Mondal et al., the ratio of obestatin/ghrelin in gastric fundus of rats was 0.004%, which is far less than what is expected in plasma.

These inconsistent results raise many interrogations regarding the exact obestatin site of production, the abundance of the peptide, and the specificity of the signal measured, as well as specific differences between humans and rodents. To gain more knowledge on obestatin, we explored its presence in different murine tissues, including the gastrointestinal tract using immunological detection in association with MS analyses in order to identify the positive immune signals. Our data confirm that both forms of ghrelin as well as obestatin are produced in majority in the gastrointestinal tract in mice.

We show that in stomach, DAG represents 50% of total ghrelin production while in plasma, it accounts for about 60% of total preproghrelin-derived peptides and nearly 80% of total ghrelin. The latter result is in accordance with previous studies using competitive immunoassays which demonstrated that DAG accounts for 80–90% of total circulating ghrelin (Hosoda et al., 2000). Moreover, we find that while plasma obestatin levels represent about 30% of all circulating preproghrelin-derived peptides, obestatin is 500–1,000 times less abundant than total ghrelin in stomach.

Although we find equimolar concentrations of total ghrelin and obestatin in mouse plasma, the amount of obestatin in tissues is negligible compared to those of ghrelin. Several hypotheses may explain this observation. First of all, conditions of sampling, processing and storage may be inadequate to preserve immunoreactive obestatin. Furthermore, a low processing rate of obestatin from preproghrelin in the stomach cannot be excluded. Finally, the existence of different transcripts arising from the preproghrelin gene in a tissue-specific manner (Seim et al., 2009), including a human transcript that encodes obestatin but not ghrelin, also suggests that obestatin transcripts may be produced independently of ghrelin.

The specificity of the immunoreactive detection for all three preproghrelin-derived peptides in both stomach and duodenum was assessed by MS and further validated by the absence of signal in preproghrelin deficient mice (Hassouna et al., 2014). Residual immunoreactivity for obestatin was detected in duodenum of two out of six preproghrelin deficient mice. This is the result of an artifact as no MS signal confirmed that it was actually obestatin. Furthermore, we also confirmed by MS that the immunoreactivity detected in tissues was specific of the amidated form of obestatin. No MS signal for all preproghrelin-derived peptides was present in stomach or duodenum of ghrl−/− mice, confirming specificity of the assay.

In this study, we used a very sensitive mass spectrometer system. Indeed detection limits is estimated to be 10–50 fmol for AG and DAG and 1–5 fmol for Obestatin-NH<sup>2</sup> and its derivatives, allowing to detect very small amounts of the peptides. The specificity was verified by determining the transitions for each peptide and analyzing ions fragments. In the original study by Zhang et al. (2005), obestatin was extracted from rat stomach but its relative abundance as compared to ghrelin was not discussed. As far as we know, this is the first attempt to identify and quantify these peptides in mouse tissues and to validate the existence of a mature form of obestatin (Obestatin-NH2) in mouse gastrointestinal tract.

In conclusion, both forms of ghrelin and obestatin can be detected with very selective immunoassays coupled with MS in gastrointestinal tract in mice. In this tissue, obestatin appears to be far less abundant than AG or DAG. This could be the result of either a lower processing rate of proghrelin into mature obestatin in gastrointestinal tissues or degradation of the peptide during the different extraction/purification procedures. Whether the main source of obestatin production and/or processing is outside the gastrointestinal tract has to be further investigated.

### AUTHOR CONTRIBUTIONS

RH: contributed to the conception and design of the work, performed experiments and analyses of data, participated to manuscript redaction. DG: contributed to reagents/materials/analysis tools, performed experiments, and analyses of data, participated to manuscript redaction. GC: contributed to the acquisition and analyses of MS data and participated to manuscript redaction. JL: contributed to the experiments and acquisition of data. OF: contributed to the experiments. CT: contributed to reagents/materials/analysis tools and revised the manuscript. JV: contributed to reagents/materials/analysis tools and revised the manuscript. JE: contributed to the conception of the work and revised the manuscript. VT: contributed to the conception and design of the work, analyses, and interpretation of data, manuscript redaction.

#### FUNDING

This work was supported by an Agence Nationale de la Recherche (ANR) Jeunes Chercheuses Jeunes Chercheurs ANR-12-JSV1- 0013-01 grant to VT, University Paris Descartes Sorbonne Paris Cité, Institut National de la Santé et de la Recherche Médicale (INSERM) and Ecole Supérieure de Physique et de Chimie Industrielles (ESPCI) Paris.

### REFERENCES

#### ACKNOWLEDGMENTS

We would like to thank Dr. Philippe Zizzari for his contribution in preparing the manuscript. We are grateful to Alice Cougnon and Chloé Got and the Plateau d'Exploration Fonctionnelle du Petit Animal in the Center for Psychiatry and Neurosciences for the care of the animals. We are grateful to Bertin Pharma (Montigny-le-Bretonneaux, France) for providing the AG and DAG assays.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnins. 2017.00211/full#supplementary-material

tissue. Biochem. Biophys. Res. Commun. 279, 909–913. doi: 10.1006/bbrc.20 00.4039

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer SF and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Hassouna, Grouselle, Chiappetta, Lipecka, Fiquet, Tomasetto, Vinh, Epelbaum and Tolle. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Development and Function of the Blood-Brain Barrier in the Context of Metabolic Control

Roberta Haddad-Tóvolli\*, Nathalia R. V. Dragano, Albina F. S. Ramalho and Licio A. Velloso\*

Laboratory of Cell Signaling and Obesity and Comorbidities Research Center, Faculty of Medical Sciences, University of Campinas, Campinas, Brazil

Under physiological conditions, the brain consumes over 20% of the whole body energy supply. The blood-brain barrier (BBB) allows dynamic interactions between blood capillaries and the neuronal network in order to provide an adequate control of molecules that are transported in and out of the brain. Alterations in the BBB structure and function affecting brain accessibility to nutrients and exit of toxins are found in a number of diseases, which in turn may disturb brain function and nutrient signaling. In this review we explore the major advances obtained in the understanding of the BBB development and how its structure impacts on function. Furthermore, we focus on the particularities of the barrier permeability in the hypothalamus, its role in metabolic control and the potential impact of hypothalamic BBB abnormities in metabolic related diseases.

#### Edited by:

Serge H. Luquet, Paris Diderot University, France

#### Reviewed by:

Fanny V. Langlet, University of Lille Nord de France, France Jacques Epelbaum, Institut National de la Santé et de la Recherche Médicale, France Miguel Lopez, Universidade de Santiago de Compostela, Spain

#### \*Correspondence:

Roberta Haddad-Tóvolli<EMAIL_ADDRESS>Licio A. Velloso<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 15 February 2017 Accepted: 04 April 2017 Published: 21 April 2017

#### Citation:

Haddad-Tóvolli R, Dragano NRV, Ramalho AFS and Velloso LA (2017) Development and Function of the Blood-Brain Barrier in the Context of Metabolic Control. Front. Neurosci. 11:224. doi: 10.3389/fnins.2017.00224 Keywords: blood-brain barrier, neurovascular unit, development, hypothalamus, inflammation, obesity

## INTRODUCTION

Blood vessels transport and deliver nutrients to warrant organism function. In the brain, they specialized into a dynamic structure that provides a protective and homeostatic interface between the central nervous system (CNS) and the remainder of the body. In this sense, the blood-brain barrier (BBB) controls the passage of selected substances into the brain, while transporting toxic products back into circulation. This efficient permeable boundary allows maintenance of the homeostasis of the CNS milieu and correct function of brain circuits.

The functionality of the BBB depends on a strict architecture. The combination of nonfenestrated brain endothelial cells (BECs) lining the walls of the CNS blood vessels, together with pericytes, neurons and glia constitute the neurovascular unit (NVU) and confer integrity to the BBB.

In the ventromedial hypothalamus, the barrier has specialized to allow the dynamic passage of hormones and nutrients from the blood to the energy-sensing arcuate nucleus of the hypothalamus (ARC) and the export of newly synthesized hormones to the pituitary. At the level of the median eminence (ME), the barrier is formed by fenestrated capillaries that allow faster transport of substances into the nutrient-sensing hypothalamic nuclei lying adjacent to it. However, ME tanycytes, specialized radial glia cells lining the walls of the third ventricle, form a physical barrier to control the correct transport of nutrients and metabolic hormones into the brain parenchyma (Weindl and Joynt, 1973; Ganong, 2000; Mullier et al., 2010; Rodríguez et al., 2010). The metabolic state, as well as the consumption of saturated fatty acids, can damage this barrier and alter the nutrient sensing of tanycytes (Lee et al., 2012; Haan et al., 2013; Langlet et al., 2013b). Changes in the development of the BBB in the hypothalamus, especially in the vicinity of the ME, may predispose to obesity (Lee et al., 2012; Kim et al., 2016).

### BBB STRUCTURE AND FUNCTION

In the last decade, there has been exponential progress in the understanding of how BBB structure and function work both in health and pathological conditions (Daneman and Prat, 2015; Banks, 2016). The first evidence for the existence of a barrier controlling the passage of substances from the circulating blood to and from the CNS dates back to 1885 when the German researcher Paul Ehrlich carried out experiments injecting Trypan Blue dye into the bloodstream of mice. Surprisingly, he noted that the dye could penetrate several tissues but not the brain and spinal cord (Ehrlich, 1885; **Figure 3B**). Years later, further studies performed by Goldmann (1909, 1913), Ehrlich's student, indicated that injection of the same dye directly into the brain showed the opposite as observed previously: the brains turned blue, whereas the peripheral tissues did not (**Figure 3B**). The term "blood-brain-barrier" was introduced by Lewandowsky (1900), based on experiments demonstrating that neurotoxic substances, e.g., cholic acids or sodium ferrocyanide, exhibited neurological symptoms only after intraventricular applications but not when injected into the bloodstream. Only in the late 1960s, with the development of electron microscopy, Reese and Karnovsky (1967) were able to visualize for the first time, at ultrastructural level, that the brain endothelial cells present unique cell-to-cell junctions, constituting a structural barrier that creates an almost impermeable frontier between the blood and CNS (Ribatti et al., 2006).

Anatomically, the BBB is comprised by a thin monolayer of BECs that are in intimate contact with vascular cells (pericytes and vascular smooth muscle cells), glial cells (astrocytes, microglia) and neurons. The crosstalk and molecular signaling between them are collectively known as the neurovascular unit (NUV) (Obermeier et al., 2013; Chow and Gu, 2015; Banks, 2016) (**Figure 1**). This close connection between different cells types within the NUV allows the BBB to properly perform its fundamental physiological functions.

Next, we present the main features of the cellular components of the BBB:

### Brain Endothelial Cells

To sustain a more restrictive permeability, BECs enclose specific barrier properties, which differentiate them from peripheral endothelial cells (Andreone et al., 2015; Chow and Gu, 2015). One particular feature is the presence of junction complexes between adjacent BECs involving transmembrane proteins and multiple cytoplasmic adaptor proteins located on the apical and lateral sides of the plasma membrane.

The structural integrity of BBB is sustained mainly by tight junction (TJ) proteins and adherens junctions (AJ). Specifically, TJ are composed of claudin family members, occludin, junctional adhesion molecules (JAMs) and zonulae occludens (ZO-1, ZO-2, and ZO-3)—membrane-associated accessory proteins that connect the cytoplasmic tails of claudins and occludin to the actin cytoskeleton to sustain the TJ structure (Luissint et al., 2012; Tietz and Engelhardt, 2015).

The primary biological role of TJs is to establish a rigorous restriction of paracellular molecular diffusion, creating a high

contact of these specialized endothelial cells with different cell types constitutes the NVU.  In areas where the basement membrane is absent, brain endothelial cells and pericytes connect through peg-socket junctions. Astrocytes extend its end-feet and establish a close interaction with endothelial cells through transmembrane proteins, such as aquaporins. Astrocytes also connect with pericytes and neurons and together regulate BBB maintenance and function. The interaction of the cell components of the NVU with neurons and microglia can influence barrier function.

electrical resistance between the endothelial cells and ions and other polar solutes. Brain endothelial cell's AJs, ubiquitous in the vasculature, consist of cadherin proteins extended throughout the intercellular cleft that mediate cell-to-cell membrane adhesion and are anchored into the actin cytoskeleton by the scaffolding proteins alpha, beta and gamma catenin (Abbott et al., 2010; Blanchette and Daneman, 2015).

Although, the specific AJs function in the BBB is yet to be fully elucidated, it is known that they play an important role in the maintenance of TJs and the junctional complex by keeping the BECs together (Keaney and Campbell, 2015). In addition, brain endothelial cells display low rates of transcellular vesicular transport in comparison to peripheral endothelial cells, a process termed transcytosis (transport between the luminal and abluminal cell membranes). Still, transcytosis is the most common mechanism to selectively uptake macromolecules such as albumin, low-density lipoprotein and hormones, e.g., insulin and leptin (Holly and Perks, 2006; Xiao and Gan, 2013; Chow and Gu, 2015). As a result of this restrictive barrier, BECs express several transporters and receptor proteins, as Glut-1, that selectively allow the entry of nutrients, neurotransmitters and other essential macromolecules into the brain parenchyma, at the same time that ensure the elimination of potentially metabolic waste and neurotoxic substances from the CNS into the blood (Keller, 2013; Keaney and Campbell, 2015).

Another important feature of the BECs is the low expression levels of leukocyte adhesion molecules (LAMs) such as Eselectin and Icam1, compared with ECs in non-neuronal tissues (Andreone et al., 2015; Daneman and Prat, 2015). This characteristic greatly restrains the number of immune cells that enter the CNS. In general, the physical barrier properties mediated by the junctional complex and the reduced transcytosis along with a highly selective cellular transport system illustrates how the brain endothelium orchestrate the regulation of the brain microenvironment.

#### Pericytes

Pericytes were first characterized by Eberth (1871) and Rouget (1873) in the 1870's and initially named "Rouget cells." Later, Zimmermann (1923) introduced the term pericyte due to their location in close proximity to the endothelial cells. Anatomically, pericytes are located at the abluminal surface of microvessels, embedded in a common basement membrane with the BECs, and exhibit elongated cytoplasmic processes that wrap the endothelium around (Trost et al., 2016). The CNS vasculature has significantly higher pericyte density/coverage compared with peripheral tissues, which correlates with the particular endothelial barrier properties. It is estimated that the ratio of pericytes to endothelial cells in CNS is 1:1-1:3, while this ratio appears to be from 1:10-1:100 in striated muscles (Shepro and Morel, 1993; Millis et al., 2013). Despite being separated by the basal membrane, in areas lacking a basement membrane (BM) BM, pericytes and BECs make direct peg-and-socket contacts, in which pericyte cytoplasmic fingers (pegs) are inserted into endothelial invaginations (pockets) (Winkler et al., 2011). Several different transmembrane junctional proteins are present at these contact points, including N-cadherin, forming key AJs between the two cell types; connexin-43 (CX43), hemichannels that form gap junctions enabling the exchange of nutrients, secondary messengers and ions; adhesion plaques composed predominately of fibronectin that anchor pericytes to endothelial cells (Berger et al., 2005; Zlokovic, 2008; Armulik et al., 2011; Winkler et al., 2011).

Pericytes play essential roles in the regulation of several neurovascular functions. They are critical during angiogenesis, participating in vessel formation, remodeling and stabilization (see developmental section) (Armulik et al., 2011; Winkler et al., 2011). In situ and In vivo studies evidenced that pericytes are modulators of capillary diameter and blood flow in response to changes in neural activity (Peppiatt et al., 2006; Fernández-Klett et al., 2010; Hall et al., 2014). However, Hill et al. (2015) demonstrate that capillary pericytes are not contractile in vivo. Thus, the active regulation of the capillary blood flow by pericytes remains controversial presumably due to the lack of proper pericyte definition and identification (Trost et al., 2016).

Apart of their importance in BBB development (see BBB development section), pericytes are involved in the formation and maintenance of the highly selected permeability of the BBB in adulthood and aging (Armulik et al., 2010; Bell et al., 2010; Daneman et al., 2010a). Pericyte deficiency promotes severe BBB dysfunction as a result of increased vascular permeability (directly correlated with absolute pericyte coverage, increased rates of transcytosis and paracellular transport due to reduced expression of various TJ and AJ proteins). Pericytes also guide astrocytic foot processes to surrounding CNS blood vessels and mediate the polarization of astrocytic end-feet, highlighting the interdependence among components of the NVU (Armulik et al., 2010; Keaney and Campbell, 2015).

Increasing evidence has shown that pericytes may constitute multipotent stem/progenitor cells that could differentiate into mesenchymal cells and into non-mesenchymal cell types, including glial and neuronal lineages under different in vitro conditions (Bribair et al., 2013). Until now, there is still a lack of studies describing that such differentiation occurs in the brain (Dore-Duffy and Cleary, 2011; Trost et al., 2016). Moreover, pericytes can control several aspects of the CNS immune response (reviewed in Rustenhoven et al., 2016).

#### Astrocytes

Due to the position of astrocytes in the NUV (ensheathing almost 90% of brain microvasculature), many studies have focused on the role of astrocytes in the maturation and maintenance of BBB (Correale and Villa, 2009). Indeed, astrocytes can induce barrier properties in non-CNS endothelial cells in vivo (Janzer and Raff, 1987).

Astrocytes, the most abundant cells in the brain, are spongiform-shaped glial cells that extend many branching cellular processes, including astrocytic end-feet that cover brain endothelial cells. Specifically, astrocytic end-feets establish a close interaction with BECs through transmembrane proteins anchoring, such as the water channel Aquaporin-4 (Aqp4) and the potassium channel KIR4.1, critical for CNS water homeostasis regulation (Cabezas et al., 2014). Furthermore, astrocytes communicate between each other through gap junctions forming an extensive glial syncytium that is associated with well-coordinated responses within large groups of cells (Theis et al., 2005; Alvarez et al., 2013). Astrocytes perform several functions critical to CNS physiology, including the regulation of blood flow in response to changes in neuronal activity which results in enhanced delivery of oxygen and glucose to the active brain region; the maintenance of ion and neurotransmitters concentration within the extracellular space for proper synaptic transmission; the modulation of synaptic and neural activity by the release of gliotransmitters, such as glutamate, ATP, D-serine, and GABA; and are also able to respond to local levels of nutrients, glucose uptake from blood vessels and the storage of glycogen, which is hydrolyzed to release lactate into the extracellular space when glucose is scarce (Sofroniew and Vinters, 2010; Cabezas et al., 2014).

One of the essential roles attributed to astrocytes is to regulate the nutrient availability, such as glucose, and also the access of some peripheral hormones, including leptin, ghrelin and GLP-1. Their privileged anatomical position, in close proximity to the blood vessels and neurons, allows them to act as important metabolic sensors. Actually, hypothalamic astrocytes express specific transporters and receptors that are involved in the control of energy homeostasis. The astrocytic end-feet that enclose capillaries express the glucose transporter GLUT-1 that transports glucose into the CNS. The uptaken glucose is stored in form of glycogen, which is mobilized to release lactate to the neurons when glucose is not abundant, as in periods of energy deficit (Argente-Arizón et al., 2015; Chowen et al., 2016). Astrocytes also express the glucose transporter GLUT-2 that is important for glucosensing process and in the homeostatic control of circulating glucose levels and food intake (Marty et al., 2005; Stolarczyk et al., 2010). GLUT-2 inactivation results in overeating, providing evidence that glucose detection by GLUT2 contributes to the control of food intake by the hypothalamus (Bady et al., 2006; Stolarczyk et al., 2010). Leptin receptors are expressed in hypothalamic astrocytes and their physiological relevance was recently demonstrated. The conditional deletion of the leptin receptor in GFAP positive cells in mice decreased the number and length of astrocyte projections in hypothalamic neurons involved in feeding control, such as POMC and AGRP neurons of the arcuate nucleus. In addition, leptin-regulated feeding was diminished in mice with astrocyte-specific leptin receptor deficiency (Kim et al., 2014).

Regarding the contribution of astrocytes in BBB development, they do not appear to participate in this process at early embryonic stages (see development section) (Daneman et al., 2010b). Indeed, mature astrocytes produce factors that regulate BBB function and integrity. Astrocytes also secrete angiopoietin-1 and angiotensin that restrict BBB permeability by supporting efficient organization of TJs (Lee et al., 2003; Wosik et al., 2007; Siegenthaler et al., 2013). Thus, astrocytes are critical in sustaining BBB functionality and integrity as well as in the regulation of neuronal and synaptic activity.

### Microglia

In addition to pericytes and astrocytes, the brain endothelium at the NUV is in close contact with immune cells. The two main immune cell populations within the CNS are perivascular macrophages and microglia.

Perivascular macrophages are monocytic lineage cells located outside of the CNS parenchyma. In fact, these cells reside in and circulate through the Virchow–Robin spaces, which lie between the BM around pericytes and at the surface of the glia interface of the brain vessels. The perivascular macrophage number is continuously maintained by replacement from blood-borne cells macrophages. They provide a first line of innate immunity due to their ability to quickly phagocytose particles from the cerebrospinal fluid (Daneman and Prat, 2015).

The brain parenchyma is populated by microglia, the most abundant CNS innate immune cells. Microglial cells, also referred as resident macrophages of the brain, are derived from progenitors in the yolk sac, which migrate into the CNS parenchyma during neonatal development. Microglia colonization comes first during BECs invasion into the brain (da Fonseca et al., 2014) and are shown to be involved in CNS vasculogenesis, promoting endothelial cell fusion to increase vascular complexity (Fantin et al., 2010), as well as decreasing paracellular permeability in cultured BECs (Zenker et al., 2003). Accordingly, these data suggest that the interactions between microglia and the brain endothelium could participate in the BBB formation and regulation (Correale and Villa, 2009; da Fonseca et al., 2014).

Generally, microglia displays two distinct morphological patterns. The steady-state population of microglia in the healthy brain exhibits a 'resting' phenotype, which is characterized by a small, and circular cell body with extensively ramified processes. As a part of their homeostatic functions, microglial cell bodies remain stationary, but their processes continuously scan the surrounding extracellular space and communicate directly with neurons, astrocytes, and blood vessels (Nayak et al., 2014). Microglia rapidly respond to injury signals or infection through an activated phenotype characterized by a morphological change into an amoeboid shape and alterations in signaling and gene expression in order to perform inflammatory functions (Lull and Block, 2010; Saijo and Glass, 2011).

Prolonged microglia activation and consequent chronic neuroinflammatory state in CNS might induce impairments in BBB integrity that, in turn, contribute to the progression of neurodegenerative diseases (reviewed in de Vries et al., 2012). Evidence from in vitro studies has shown that microglia activation may be related to BBB disruption. Sumi et al. (2010) demonstrated that the treatment of a rat brain microvascular endothelial cell/microglia co-culture system with lipopolysaccharide, a microglial activator, induced an increase in endothelial cell permeability and changes in the expression pattern of tight junction proteins. Another study using a similar in vitro co-culture system found that the tumoral necrosis factor α (TNF-α) released from activated microglia also increased the permeability of BECs, which could be blocked by a neutralizing antibody against TNF-α (Nishioku et al., 2010).

Although, these in vitro BBB models are important for unraveling the mechanisms involved in the crosstalk between microglia activation and BBB integrity, more studies are needed to improve our understanding on how these cell types interact in vivo.

### BBB DEVELOPMENT

The development and differentiation of the BBB can be subdivided into three phases: i, angiogenesis; ii, differentiation; and iii, maturation (summarized in **Figure 2**).

The angiogenic phase begins early during neural tube development (as early as E9.0-E10.5 in mice).  VEGF is expressed by the neural progenitors of the ventricular neuroepithelium and serves, together with angiopoietin, as a major driving force for the migrating endothelial cells (EC) (Risau et al., 1986). VEGF deficiency is lethal (Shalaby et al., 1995; Carmeliet et al., 1996).

penetration into the brain parenchyma according to a VEGF concentration gradient. Wnt ligands also secreted from the NPCs induces the migration of the endothelial cells and activate β-catenin signaling through the binding to Frizzled receptors, inducing the expression of BBB specific genes. GPR124, together with WNT, co-activates β-catenin signaling. Endothelial cells secrete PDGF-B and attract pericytes expressing PDGFR-β. The interaction between ECs and Ps induce the mutual expression of TGF-β and TGF-βR2. The activation of TGF-β signaling regulates basement membrane formation and the induction of Ang-1 expression in pericytes, that acting through the endothelial receptor Tie-2, enhance tight junction expression. Astrocytes release SHH, that when bound to PTCH receptor induces Shh signaling activation in ECs and contributes to BBB formation. Astrocytes also express ANT and ANG-1, which by limiting BBB permeability contributes to the maturation of BBB function. (B) Maturation and Maintenance. Pericytes and astrocytic end-feet cover the endothelium and secrete matrix proteins that will constitute the basement membrane. Astrocytes continue secreting SHH and WNT in order to maintain BBB functionality throughout life. A, astrocytes; E, endothelium; EC, endothelial cell; M, microglia; N, neuron; NPC, neural progenitor cell; P, pericyte; TJ, tight junction; Ang-1, angiopoietin-1; ANT, angiotensin; β-cat, beta-catenin; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; FZD, frizzled; GPR124, adhesion G protein coupled receptor A2; Shh, sonic hedgehog; Ptch, Patched 1; PDGF-B, platelet derived growth factor B; PDGFR-β, platelet derived growth factor receptor beta; TGF-β, transforming growth factor-beta; TGF-βR2, transforming growth factor beta receptor type-2.

Downstream VEGF signaling supports angiogenesis through endothelial cell proliferation, migration and survival (Olsson et al., 2006). Together with VEGF, neural Wnt signaling plays an important role in the development of the BBB (Daneman et al., 2009). Different Wnt ligands, including Wnt7a, Wnt7b, and Wnt3a are secreted by the neuroepithelium (Wang et al., 2012) and induce further ingression of ECs into the neural tissue, activating Wnt/B-catenin signaling in the newborn endothelial cells (Liebner et al., 2008; Daneman et al., 2009; Zhou and Nathans, 2014) that leads to induction of genes critical for the BBB formation and vascular patterning, such as glucose transporter Glut-1 (Stenman et al., 2008), death receptors DR6 and TROY (Tam et al., 2012) and tight junction proteins. Defects in Wnt/β-catenin signaling result in major vascular malformations and BBB breakdown (Stenman et al., 2008; Daneman et al., 2009; Wang et al., 2012). Retinoic acid, Notch signaling receptor tyrosine kinase, cadherins and ephrins also contribute to angiogenesis in the CNS (Adams and Alitalo, 2007; Dejana and Vestweber, 2013).

At about E15.5, the newly formed vessels continue to differentiate and mature as a complete NVU forming the basis for the BBB. During the differentiation phase (between E15.5 and E18.5 in mice), the brain barrier is structured properly by the induction of anti-angiogenic signals and the recruitment of pericytes and astrocytes to the newly formed vessels. Pericytes express the platelet-derived growth factor receptor- β (Pdgfr-β) and are directed to the endothelial developing capillaries that secrete Pdgf-B (Lindahl et al., 1997; Hellstrom et al., 1999).  The lack of Pdgf-β or Pdgfr-β leads to erroneous TJ distribution and increased vascular permeability (Hellstrom et al., 2001).

Interactions between pericytes and the brain endothelial cells lead to the expression of transforming growth factor-β (Tgf-β) and its receptor (Tgf-βR2) by both cell types. TGF secretion induces cell adhesion through the production of cadherin-2 by the endothelial cells and the secretion of different extracellular matrix components that contribute to basement membrane (BM) formation by the pericytes (Winkler et al., 2011). Notch and sphingosine-1-phosphate (S1P) signaling also contribute to the regulation of cadherin-2 expression in BECs (Winkler et al., 2011; Obermeier et al., 2013). Upon activation of Tgf-β signaling, pericytes produce Ang-1 that, by enhancing tight junctions formation, limit BBB permeability, and reduce the expression of leukocyte adhesion molecules (LAMs). Pdgf-β and Wnt/βcatenin signaling is also important during the differentiation of the BBB by the induction of transporters and increased expression of tight junctions. This provides integrity to the barrier (Liebner et al., 2008; Daneman et al., 2009; Tam et al., 2012; Wang et al., 2012; Zhou and Nathans, 2014; Andreone et al., 2015). In the BBB, Gpr124 has been described as a necessary endothelial receptor specifically in the CNS and function as a coactivator of Wnt/b-catenin signaling to mature the BBB (Kuhnert et al., 2010; Cullen et al., 2011; Zhou and Nathans, 2014).

Pericytes are apparently required to guide astrocytes toward the developing BBB (Armulik et al., 2010). Once recruited to the forming NVU, astrocytes are involved in limiting BBB permeability by the release of Sonic Hedgehog (Shh). The activation of Shh signaling leads to the expression of occludin and claudin5 and inhibition of chemokines and cell adhesion molecules in the endothelial cells, suggesting a role for astrocytes and Shh in maintenance of BBB functionality and immune surveillance (Alvarez et al., 2011; Obermeier et al., 2013; Siegenthaler et al., 2013). Recent studies have uncovered yet another important role for astrocytes in the production of retinoic acid, which is also necessary for the correct development of BBB vessels (Halilagic et al., 2007; Mizee et al., 2013).

Maturation and maintenance of the BBB is achieved by the persistence of tight junction proteins expression and their redistribution throughout the whole BBB structure. The production of TJs is regulated by Wnt signaling between astrocytes and BECs. Close contact between the endothelial cells, pericytes, astrocytes, and possibly neurons and microglia sustain BBB integrity and function as a stabilized neurovascular unit (reviewed in Obermeier et al., 2013; reviewed in Engelhardt and Liebner, 2014; reviewed in Zhao et al., 2015). The BBB is already formed and completely functional during late gestation in rodents and in the third trimester in humans (Bauer et al., 1995; Daneman et al., 2010a; Ben-Zvi et al., 2014). In the rat embryo, the BBB is already functional at E16 (Saunders et al., 2012). The exact time window remains elusive and is likely species dependent (Saunders et al., 2013; Hagan and Ben-Zvi, 2015).

There has been intense debate on the BBB features that provide its correct function. To date, it is believed that tight junctions are functional in sealing the space between BBB endothelial cells very early in development (Dziegielewska et al., 1979; Bondjers et al., 2006; Tam et al., 2012; Ben-Zvi et al., 2014) but only when the intracellular pathway of transcytosis is partially downregulated will the complete restrictive properties of the barrier become fully matured (Hagan and Ben-Zvi, 2015). Little is known about the functionality of transporters at the embryonic BBB, although some transporters, like Glut-1, are known to be expressed very early (Boado and Pardridge, 1990; Farrell and Pardridge, 1991; Bauer et al., 1995; Hagan and Ben-Zvi, 2015). A role of Mfsd2a in participating in the inhibition of vesicular trafficking and inducing a more sealed barrier during development was suggested recently (Ben-Zvi et al., 2014; reviewed in Hagan and Ben-Zvi, 2015). Mfsd2a mutants have a defective BBB even though presenting proper and functional tight junctions (Ben-Zvi et al., 2014). Thus, barrier transporting properties would be determined very early in development while the sealing function would be acquired gradually across development, first with the suppression of fenestrations, then with the appearance of functional TJs, and finally with the decrease of transcytosis by the expression of Mfsd2 (Ben-Zvi et al., 2014; Hagan and Ben-Zvi, 2015).

Alterations in barrier development and in TJ expression could lead to anomalies later in life as well as to increased predisposition to develop metabolic diseases. Maternal obesity increases BBB permeability in the offspring (Kim et al., 2016), leading to higher exposure to leptin and ghrelin. In consistence with that, overnutrition during early postnatal life alters brain sensitivity to ghrelin (Collden et al., 2014), suggesting that nutrient sensing control alterations during specific hypothalamic developmental time-points could contribute to metabolic defects in the adult life. These alterations can affect the programming of energy homeostasis circuits predisposing the offspring to the development of metabolic syndrome at early life and/or adulthood.

### BBB IN THE HYPOTHALAMIC AREA

In some areas of the brain, the BBB is modified in order to allow the access of certain substances from systemic circulation into the central nervous system. This occurs particularly in periventricular areas creating a blood/spinal fluid interface (Bennett et al., 2009). In these regions, the BBB capillaries are highly fenestrated with less tight junctions between endothelial cells creating a more permeable barrier (Bennett et al., 2009). Seven periventricular regions display differential barrier properties and are collectively known as the circumventricular organs: i, sub-fornical organ; ii, organum vasculosum of the lamina terminalis; iii, pituitary; iv, area postrema; v, median eminence (ME); vi, subcomissural organ; and vii, pineal (Bennett et al., 2009; Szathmari et al., 2013). In these regions, in addition to the cells that classically form the BBB vessels, a differential type of radial glial cell, termed tanycytes are also found in the interface between the spinal fluid and the capillaries (Rodríguez et al., 2010). Because of its physical proximity with the hypothalamus, the BBB at the ME is of particular interest regarding whole-body energy homeostasis and metabolism.

The median eminence is adjacent to the arcuate nucleus (ARC) of the hypothalamus. This region harbors neurons that play important roles in the control of whole body metabolism (Velloso and Schwartz, 2011; Cavadas et al., 2016). Classically, two main neuronal populations control energy homeostasis in the ARC: i, NPY/AgRP neurons, which are active during fasting and provide orexigenic and anti-thermogenic signals; ii, POMC/CART neurons, which are active following food intake and provide anorexigenic and pro-thermogenic signals (Velloso and Schwartz, 2011; Cavadas et al., 2016). In addition,

FIGURE 3 | Organization of the BBB in the energy-sensing hypothalamus. (A) Coronal section of the tuberal hypothalamus showing the distribution of tanycytes along the third ventricle wall. Left: vimentin staining (red) shows the projections of tanycytes to the brain parenchyma. Right: tanycytes line the third ventricle and can be classified according to location and function. α tanycytes don't possess barrier properties. α1 tanycytes (dark red) lye in the dorsal ventro-medial nucleus of the hypothalamus, while α2 tanycytes (light red) are found in between the ARC and the VMH. β tanycytes are located ventrally and function as gatekeeper cells controlling the passage of substances from the leaky ME to the ARC. β1 tanycytes (turquose) divide the ME from the ARC, while β2 tanycytes (green) are located in the ME, and characterized by processes with direct access to the blood capillaries. Blood capillaries are displayed in light pink.  On the other hand, dyes that are infused inside of the brain ventricles diffuse trhough the CSF and penetrate the brain parenchyma but do not pass the ME in the direction of the portal capillaries (right). VMN, ventro-medial nucleus; ARC, arcuate nucleus; ME, median eminence; 3V, third ventricle.

other distinct hypothalamic neuronal subpopulations have been described recently (Zhang and van den Pol, 2016; Campbell et al., 2017; Fenselau et al., 2017; Lam et al., 2017). Of note, a recent study has identified ARC neurons that express the dopaminesynthesizing enzyme, tyrosine hydroxylase (TH). These cells are involved in the integration of homeostatic and hedonic feeding signals and are potential targets for the treatment of obesity (Zhang and van den Pol, 2016).

Because ARC neurons are involved in the sensing of the nutritional status of the body, it would be expected that they were located in an anatomical area where the access to nutrients and energy-status signaling substances would be facilitated. In fact, studies have shown that the interface between the median eminence and the ARC is somewhat leaky to hormones and nutrients (Obici and Rossetti, 2003; Lam et al., 2005). For example, fatty acids present in the systemic circulation are not freely diffusible to most areas of the CNS (Mitchell and Hatch, 2011). In general, they rely on particular transport systems present in the structures of the BBB (Betsholtz, 2014). The proper function and distribution of these transport systems is of major importance in the development and physiology of the brain throughout life because most fatty acids that comprise the central nervous system phospholipids cannot be synthesized de novo in the brain, and thus, must be obtained from the systemic circulation (Mitchell and Hatch, 2011). However, in the ME specific conditions make circulating fatty acids more available to ARC neurons (Obici and Rossetti, 2003; Lam et al., 2005).

As early as the 1970's, studies have revealed that hormones involved in energy homeostasis and nutrients have particular properties to cross the BBB at the ME/ARC interface. Systemic insulin crosses the BBB by a saturable system (Woods and Porte, 1977) and concentrates mostly in the olfactory bulb and hypothalamus (Havrankova et al., 1981). Similarly, leptin reaches the brain by a saturable system, which is independent of insulin and is highly detectable in the choroid plexus, the ME and the ARC (Banks et al., 1996). A major advance in understanding how hypothalamic neurons are exposed to hormones and nutrients to respond to systemic variations in whole body energy status was obtained by the characterization of the presence of nutrient transporters in BBB endothelial cells. Beginning in the 1990's a series of studies have explored the particularities of how peptides can cross the BBB (Cashion et al., 1996; Pam et al., 1996; Banks et al., 1997). The BBB endothelial cells are also provided with transport systems for glucose (Pardridge et al., 1990); fatty acids (Obici and Rossetti, 2003; Lam et al., 2005) and aminoacids (Hawkins et al., 2006), and the variations in whole body energy status, such as in fasting/feeding or metabolic diseases can affect function of such transport systems (Kastin and Akerstrom, 2000; Kastin et al., 2001). This implies that the BBB at the ME/ARC interface plays a major role in controlling the way hypothalamic neurons are exposed to systemic factors involved in metabolism and nutrition. In this context, important advance in the field by exploring the mechanism involved in the regulation of the ME tanycytes has been provided over the last few years.

Tanycytes are originated from the radial bipolar glia around E17.5 in mice and have a morphology that is somewhere in between the aspects of an ependymal cell and an astrocyte; differing from the last by having a single basal projection directed toward the parenchyma of the brain (Rodríguez et al., 2010; **Figure 3A**).

Mounting evidence suggests that, at least part of the selective leakiness of the BBB at the median eminence relies on the responsiveness of tanycytes to nutrients present in the bloodstream. Early studies evaluating hypothalamic tanycytes have demonstrated their role in the transport of substances between the third ventricle and ME (Wagner and Pilgrim, 1974). Also, tanycytes were shown to respond to hormone production and direct hormone delivery to certain anatomical sites and systemic circulation (Akmayev and Popov, 1977; Vallet et al., 1991). Interestingly, tanycytes respond rapidly to systemic stimulation, suggesting that this particular cell-type could play a role in the dynamic control of median eminence and adjacent areas exposure to systemic factors (Lichtensteiger et al., 1978). Recent studies have provided important progress in the understanding of the roles played by tanycytes in the ME and ARC (Langlet et al., 2013a,b).

The identification of leptin in the mid 1990's placed the ARC in the center of a complex system that controls body energy status (Zhang et al., 1994). With this concept in mind, researchers looked for a potential involvement of ME tanycytes as gatekeepers controlling the access of substances (particularly nutrients and hormones) that could modulate ARC-neuron function. However, it was only in 2011 that first evidence was provided showing that tanycytes are responsive to glucose fluctuations promoted by feeding (Frayling et al., 2011). Glucose leads to a powerful ATP-mediated Ca2<sup>+</sup> release into the tanycytes, which in turn release ATP to adjacent cells. Therefore, it was proposed that by responding to glucose and releasing ATP, ME tanycytes could modulate the activity of ARC neurons (Frayling et al., 2011). Furthermore, ME tanycytes can transport leptin into the mediobasal hypothalamus (Balland et al., 2014). When leptin present in the bloodstream reaches the median eminence it activates leptin receptors expressed in the tanycytes. This in turn, activates the protein ERK that transduces the signals required for an appropriate transport of leptin through the cerebrospinal fluid into the ARC (Balland et al., 2014).

Interestingly, in order to respond to the constant changes in nutrient and hormone availability in the circulation, the plasticity of the median eminence tanycytes has proven remarkable. During the physiological cycles of fasting and feeding these cells undergo both morphological and functional changes (Langlet et al., 2013a). The decreased blood levels of glucose during fasting are capable of inducing changes in the structure of the interface between the blood and the ARC. At least in part, these morphological and functional changes are dependent of the expression of VEGF-A by the tanycytes (Langlet et al., 2013a).

In addition to glucose and leptin, fatty acids can also affect ME tanycytes. In diet-induced obesity, the amount of lipid

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droplets increase considerably in tanycytes (Hofmann et al., 2017). Moreover, the ratio between saturated and unsaturated lipids is modified suggesting that this cell type may also act as a sensor and gatekeeper for lipids in the median eminence/ARC interface (Hofmann et al., 2017).

#### CONCLUDING REMARKS

The BBB provides a physiological interface between the central nervous system and the systemic circulation allowing the entrance of nutrients and certain signaling molecules and restraining the entrance of microorganisms and particles that can harm the brain. Because hypothalamic neurons must sense the energy status of the body, the BBB at the median eminence specialized to a more permeable interface between the vasculature and the brain. Recent studies have demonstrated that tanycytes present in the interface between the ARC and the median eminence are very sensitive to nutrients. They can be rapidly modified in response to fast and fed states and also, can be disturbed by abnormal consumption of nutrients derived from the diet. In obesity and in metabolic conditions associated with the obese phenotype, hypothalamic neurons are affected by a local inflammatory response that is triggered by the excessive amount of fatty acids in the diet. Mounting evidence suggest that, at least in part, the anomalous activity of the hypothalamic tanycytes can play a role in the defective neuronal activity in obesity and associated conditions. Future studies should focus on the identification of mechanisms that may protect the tanycytes from diet-induced abnormalities and the impact of such protection in the progression of metabolic diseases.

#### AUTHOR CONTRIBUTIONS

RHT, NRVD, and LAV discussed the structure of the manuscript. NRVD wrote the aspects of the BBB structure and function and RHT wrote the developmental topic. LAV and AFSR wrote BBB specialization in the hypothalamus. RHT, NRVD, and LAV wrote the introduction, abstract, and conclusion remarks. RHT made the figures and figure legends.

### ACKNOWLEDGMENTS

The writing of this article was supported by São Paulo Research Foundation (2013/07607-8 and 2015/50278-0). Funding was provided by São Paulo Research Foundation as postdoctoral fellowships to RHT (2015/02913-9 and 2016/01868-2) and NRVD (2014/26942-5). The Laboratory of Cell Signaling belongs to the Obesity and Comorbidities Research Center and the National Institute of Science and Technology—Diabetes and Obesity.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Haddad-Tóvolli, Dragano, Ramalho and Velloso. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effects of Fat and Sugar, Either Consumed or Infused toward the Brain, on Hypothalamic ER Stress Markers

Evita Belegri <sup>1</sup> , Merel Rijnsburger <sup>1</sup> , Leslie Eggels <sup>1</sup> , Unga Unmehopa<sup>1</sup> , Wiep Scheper <sup>2</sup> , Anita Boelen1 † and Susanne E. la Fleur 1, 3 \* †

<sup>1</sup> Laboratory of Endocrinology, Department of Clinical Chemistry and Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands, <sup>2</sup> Clincal Genetics, VU Medical Center, Amsterdam, Netherlands, <sup>3</sup> Metabolism and Reward Group, Netherlands Institute for Neuroscience, Amsterdam, Netherlands

#### Edited by:

Hubert Vaudry, University of Rouen, France

#### Reviewed by:

Denis Richard, Laval University, Canada Julie A. Chowen, Hospital Infantil Universitario Niño Jesús, Spain

> \*Correspondence: Susanne E. la Fleur

<EMAIL_ADDRESS>
These authors have contributed equally to this work.

†

#### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 15 February 2017 Accepted: 26 April 2017 Published: 15 May 2017

#### Citation:

Belegri E, Rijnsburger M, Eggels L, Unmehopa U, Scheper W, Boelen A and la Fleur SE (2017) Effects of Fat and Sugar, Either Consumed or Infused toward the Brain, on Hypothalamic ER Stress Markers. Front. Neurosci. 11:270. doi: 10.3389/fnins.2017.00270 Protein-folding stress at the Endoplasmic Reticulum (ER) occurs in the hypothalamus during diet-induced obesity (DIO) and is linked to metabolic disease development. ER stress is buffered by the activation of the unfolded protein response (UPR), a controlled network of pathways inducing a set of genes that recovers ER function. However, it is unclear whether hypothalamic ER stress during DIO results from obesity related changes or from direct nutrient effects in the brain. We here investigated mRNA expression of UPR markers in the hypothalamus of rats that were exposed to a free choice high-fat high-sugar (fcHFHS) diet for 1 week and then overnight fed ad libitum, or fasted, or fat/sugar deprived (i.e., switched from obesogenic diet to chow). In addition, we determined the direct effects of fat/sugar on mRNA expression of hypothalamus UPR markers by intracarotic infusions of intralipids and/or glucose in chow-fed rats that were fasted overnight. Short term (1 week) exposure to fcHFHS diet increased adiposity compared to chow-feeding. Short term exposure to a fcHFHS diet, followed by mild food restriction overnight, induced hypothalamic ER stress in rats as characterized by an increase in spliced to unspliced X-box binding protein 1 mRNA ratio in hypothalamus of fcHFHS fed rats compared to chow fed rats. Moreover, infused lipids toward the brain of overnight fasted rats, were able to induce a similar response. Non-restricted ad libitum fcHFHS-diet fed or totally fasted rats did not show altered ratios. We also observed a clear increase in hypothalamic activating transcription factor 4 mRNA in rats on the fcHFHS diet while being ad libitum fed or when infused with intralipid via the carotic artery compared to vehicle infusions. However, we did not observe induction of downstream targets implying that this effect is a more general stress response and not related to ER stress. Overall, we conclude that the hypothalamic stress response might be a sensitive sensor of fat and energy status.

Keywords: ER stress response, hypothalamus, food restriction, fatty acids, sugar

## INTRODUCTION

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 proopiomelanocortin (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.

### MATERIALS AND METHODS

#### Animals

Adult male Wistar rats (250–280 g, Charles River, Germany) were individually housed in a temperature controlled room (19 ± 1 ◦C) on a 12 h light/ 12 h dark cycle (lights on at 7:00 a.m.). During the experiments animals had ad libitum access to water and standard laboratory chow (SDS, UK) unless stated differently. All the studies were approved by and performed according to the regulations of the Committee for Animal Experimentation of the Academic Medical Centre of the University of Amsterdam, Netherlands.

### fcHFHS Diet Experiments

Three experiments were performed to study the effect of nutrient availability on the hypothalamic ER stress response and its interaction with obesity. In all experiments rats were on a fcHFHS diet or chow for 1 week. The fcHFHS diet consisted of ad libitum access to chow, tap water, 30% sugar water (1.0 M sucrose mixed from commercial grade sugar and water) and a dish of pure animal fat (beef tallow; Ossewit/Blanc de Boeuf, Vandermoortele, Belgium) (composition: 34% oleic acid, 25% plamitc, and 22% stearic acid and 4% linoleic acid). After this period the three experiments differed in feeding regime the night before the end of the experiment (4:00 p.m. day 7–9:00 a.m. day 8).

Fat/sugar deprivation in our diet model is characterized by removing the fat/sugar components of the diet (i.e., saturated fat and sugar water) overnight. We previously showed that removing fat and sugar from the fcHFHS diet results in consumption of 10–15 g chow spontaneously without caloric compensation for the fat/sugar components of the diet (Pandit, 2015). To ensure equal intake overnight between fcHFHS and chow-fed rats we provided all animals with 10 g of chow. Food components of the diet were weighed 5 times a week and the amounts of components eaten were multiplied with the caloric value of each component to determine energy intake in kcals.

At the end of the experiment, rats were anesthetized via 30% CO2/70% O<sup>2</sup> and decapitated between 9:00 a.m. and 11:00 a.m. Epididymal, mesenteric, subcutaneous and peritoneal fat were dissected and weighed, trunk blood collected and brains were removed and stored at −80◦C until further analysis. Plasma leptin concentrations were determined by radioimmunoassay

Source; Fresenius Kabi.

TABLE 2 | Primer sequences used for RT-PCR.

(Linco Research, Inc., St. Charles, MO, USA). Samples were assayed in duplicate. Amounts of sample, standards, label, antibody and precipitating reagent as described in the procedures of the assay were divided by 4. The detection limit was 0.5 ng/ml and the inter- and intra-assay coefficients were 8% or less.

### Intracarotic Infusion Experiment

Rats (n = 6–9 per group) underwent surgery under anesthesia induced with an i.p. injection of 80 mg/kg Ketamin (Eurovet Animal Health, Netherlands), 8 mg/kg Xylazin (Bayer Health Care) and 0.1 mg/kg Atropin (Pharmachemie, Netherlands). A silicon catheter was inserted in the carotid artery and directed toward the brain (according to the method of Steffens, 1969). The catheter was externalized at the vertex of the head and the animals were allowed to recover for 7 days.

To study the direct effect of fat and/or glucose on hypothalamic ER stress markers, NaCl (control), 20% IL [(Fresenuis Kabi); composition of IL is displayed in **Table 1**] or IL + 1% glucose (G) were infused via the carotid artery toward the brain (experiment 4). Another infusion experiment was performed to study the effect of glucose on hypothalamic ER stress response using glucose (1% in NaCl) or NaCl (experiment 5). All solutions were heparinized (0.04%) and Infusion rate was 5µl/min for 2 h. One hour after the end of infusion the animals received a single shot of pentobarbital via the carotid artery (100–150 mg/kg BW) and were decapitated. Brains were removed and stored at −80◦C for further analysis.

### Brain Harvesting—Isolation of the Hypothalamus

Coronal brain slices of 250µm were obtained from −0.96 to −4.36 mm Bregma (Rat brain atlas; Paxinos and Watson, 2007) and directly put in RNAlater solution (Ambion Life Technologies). The hypothalamic part in each section was isolated using syringe needles (0.4 × 19 mm, BD Microlance) and used for RT-PCR.

#### RNA Isolation—RT-PCR

One half of the hypothalamus was homogenized in lysis buffer provided with the "High Pure RNA isolation kit" (Roche

\*Oslowski and Urano (2011).

Molecular Biochemicals, Manheim, Germany) and total RNA was isolated according to the manufacturer's instructions. RNA was quantified by spectrophotometry at 260 nm (Nanodrop 1000, Willmington, Delaware, USA) and cDNA synthesis was performed using the "Transcriptor First Strand cDNA synthesis kit" for RT PCR with oligo(dT) primers (Roche Molecular Biochemicals, Manheim, Germany). The mRNA levels of ER stress markers as well as Hypoxanthine-guanine phosphoribosyltransferase (Hprt), Cyclophilin A and β-actin were determined by RT-PCR using SensiFAST SYBR No-Rox mix (Bioline, Luckenwalde, Germany) at the Lightcycler 480 apparatus (Roche Molecular Biochemicals, Manheim, Germany). The primers were designed using "Primer Blast" (**Table 2**). Samples were baseline corrected and individually checked for their PCR efficiency using the "LC480 Conversion" and "LinRegPCR" software. Median efficiency was calculated for each assay and samples that differed more than 0.05 from the mean efficiency were excluded from statistical analysis. Specific gene expression was normalized to the geometric mean of three housekeeping genes; (Hprt × β-actin × Cyclophilin A)1/<sup>3</sup> .

### Statistical Analysis

Data are presented as Mean ± SEM. Outliers were detected using "Dixon's Q test" and excluded. Differences between diet groups were evaluated using Student's t-test [experiments 1–4, 5 (NaCl vs. G)] or ANOVA followed by post-hoc Tukey test (experiment 5, NaCl vs. IL vs. IL+G). Difference between groups was considered significant when p < 0.05. In order to determine the effects of low vs. high fat intake and low vs. high sugar intake a median split was performed whereby the median was calculated for fat or for sugar intake and the animals that consumed more than the median were depicted as high consumers and those that consumed lower as low consumers. All tests were performed using Graphpad Prism 6 (Graphpad software Inc., la Jolla, CA, USA).

### RESULTS

### One Week of fcHFHS Diet Does Not Induce Hypothalamic ER Stress Markers

Rats exposed to the fcHFHS diet for 1 week were hyperphagic as shown by increased caloric intake compared to those on chow (**Table 3**). As a result, % WAT/BW and plasma leptin levels were significantly higher in rats on the fcHFHS diet compared to rats on chow whereas 1BW did not differ between the groups (**Table 3**).

One week of fcHFHS diet exposure significantly increased ATF4 mRNA expression (**Figure 1A**). However, mRNA expression of CHOP, a target gene of ATF4, and BiP, a target gene of ATF6, was not significantly changed in hypothalami of fcHFHS-fed rats compared to chow-fed controls (**Figures 1B,C**). Splicing of usXBP1 to sXBP1 as shown by the ratio sXBP1/usXBP1 (**Figure 1D**), as well as DP5 and FasL mRNA expression did not differ between the groups.

### Overnight Fat/Sugar Deprivation Induces ER Stress in Animals Exposed to the fcHFHS Diet for 1 Week

1BW, % WAT/BW and leptin were still increased in fcHFHSfed rats compared to chow-fed rats when overnight deprived of fat and sugar (**Table 3**), but no changes were observed in hypothalamic ATF4, CHOP, and BiP mRNA expression between the groups (**Figures 2A–C**; left column). However, sXBP1/usXBP1 mRNA was higher and DP5 mRNA was lower in the fcHFHS-fed group compared to the chow controls (**Figures 2D,E**; left column).

FIGURE 1 | ER stress markers in rat hypothalamus after 1 week ad libitum fcHFHS diet or chow. (A) ATF4 and (B) CHOP (C) BiP, and (D) sXBP1/usXBP1 mRNA expression. mRNA expression of specific genes was normalized to the geometric mean of three housekeeping genes. Significant differences between the fcHFHS and control group: \*p < 0.05.

#### TABLE 3 | Characteristics of chow and fcHFHS animals.

Delta body weight, average food intake per day, % total white adipose tissue (WAT) relative to final body weight of animals and leptin plasma concentrations in experiment 1 (ad lib fed o/n), 2 (10 g chow fed o/n) and experiment 3 (fasted o/n) are shown as mean (n = 8) ± SEM. Total WAT represents the sum of mesenteric, peritoneal, subcutaneous and epididymal fat. Significant differences between the fcHFHS and chow control group: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001.

No change in FasL mRNA expression was observed between the groups after removing fat and sugar overnight (not shown). Similarly, no differences in ATF4, CHOP, BiP, and FasL mRNA expression were observed between the groups after overnight fasting (**Figure 2**, right column). sXBP1/usXBP1 and DP5 mRNA expression were not detectable in the overnight fasted groups.

### Fat Consumption Leads the Changes Induced in Stress Gene Markers

To determine to what extent fat or sugar contributed to the observed differences observed in hypothalamic mRNA, we divided the fcHFHS group according to fat and sugar intake using a median split forming either low or high fat consumers (LF or HF) or low or high sugar consumers (LS or HS). The median fat consumption was 10% out of total caloric intake. Rats that consumed <10% fat were assigned as LF consumers and those that consumed >10% as HF consumers. LF and HF consumers had similar BW, total caloric intake and chow intake, but fat consumption in HF group was significantly higher and sugar intake significantly lower compared to the LF group (**Table 4**). ATF4 mRNA expression tended to be higher (p = 0.07), in the hypothalamus of the HF consumers compared to the LF consumers, whereas sXBP1/usXBP1, DP5 and FasL mRNA expression was not different between the groups (data not shown). Similar analysis was performed for consumption of sugar over a week of fcHFHS exposure and median sugar consumption out of total caloric intake was 38%. No differences in ATF4, usXBP1, sXBP1/usXBP1, DP5, or FasL mRNA expression were observed between HS and LS consumers (data not shown).

To further investigate the direct effects of fat and sugar in the brain on hypothalamic ER stress markers, we infused IL, IL + glucose (ILG), glucose or saline (control) via the carotid artery directly to the brain of overnight fasted rats. IL and ILG increased hypothalamic ATF4 mRNA expression compared to saline but did not affect CHOP, DP5, and FasL mRNA (**Figures 3A–D**). In addition, IL infusion resulted in increased sXBP1/usXBP1 mRNA expression, and tended to increase BiP mRNA expression (**Figures 3E,F**). Glucose infusion did not have an effect on AFT4, sXBP1/usXBP1, and DP5 mRNA expression (**Figures 3G–I,K**), but reduced BiP and FasL mRNA levels compared to saline infusion (**Figures 3J,L**).

#### DISCUSSION

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

Average Total, Chow, Fat, or Sugar caloric intake as well as body weight and % Fat/BW of animals over 1 week on fcHFHS diet.  The median of fat intake was 10% out of total caloric intake. Significant differences between the HF and LF group: \*\*p < 0.01.

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.