Synaptotagmin 4: A New Anti-obesity Target?

Abstract

In this issue of Neuron, Zhang et al. show that Synaptotagmin 4 is specifically induced in adult hypothalamic oxytocin neurons by high-fat diet. Evidence is provided to support a critical role for Synaptotagmin 4 in negative regulation of oxytocin release, which in turn is responsible for diet-induced obesity, raising the possibility of using Synaptotagmin 4 as a new anti-obesity target.

Given the increasing prevalence of obesity and the devastating co-morbidities associated with obesity, identifying effective anti-obesity strategies is more imperative than ever. Although the underlying causes of the obesity epidemic are multi-factorial, exposure to high-caloric diet (western diet) is thought to be one of the major reasons. In order to mimic human obesity in animal models, a widely accepted strategy involves inducing obesity in rodent models with high-fat diet (HFD) feeding. The HFD feeding can induce obesity and metabolic disorders in rodents that resemble the human metabolic syndrome (Buettner et al., 2007). Thus, important anti-obesity drug targets can be identified using HFD-induced obesity models.

Research efforts in the last decades have established that the hypothalamus plays a central role in body weight regulation. The hypothalamus contains diverse groups of body weight-regulating neurons that release distinct neurotransmitters, the most studied of which are neuropeptides (Elmquist et al., 2005). Recent evidence suggests that the oxytocin-releasing neurons, located in the paraventricular hypothalamus (PVH) and the supraoptic nucleus, are implicated in body weight regulation, in addition to their well established role in social cognition (Donaldson and Young, 2008). Reduced oxytocin expression has been associated with mouse models of obesity (Kublaoui et al., 2008); pharmacological studies demonstrate that oxytocin inhibits feeding involving a projection from the PVH to the hindbrain, where meal size is regulated (Blevins et al., 2004); and anatomically, oxytocin neurons express important body weight regulators including Fto, a prominent obesity-associated gene identified in human genetic studies (Olszewski et al., 2009). However, it is unknown whether dysfunction in oxytocin neurons contributes to the pathogenesis of HFD-induced obesity.

Although the mechanisms underlying hypothalamic neuropeptides in the regulation of body weight remain to be established, it has been commonly thought that the rate of neuropeptide release correlates with the expression level of the peptide and with neuronal activity. This is because neurons are equipped with sophisticated but relatively rigid secretory machinery that is responsible for a series of events, which lead to neurotransmitter release (Pang and Sudhof, 2010). One key event in this process involves Ca²⁺ triggered vesicle fusion with the presynaptic membrane mediated by the fusion complex. Synaptotagmin, a key component of the fusion complex, senses Ca²⁺ and triggers final fusion pore formation (Pang and Sudhof, 2010). Interestingly, within the synaptotagmin family, the mammalian synaptotagmin 4 (Syt4) is atypical in that it is insensitive to Ca²⁺, but still capable of participating in the fusion complex. Although controversy exists regarding the action of Syt4 on synaptic release, strong data support that Syt 4 reduces synaptic release, presumably by decreasing the frequency of fusion events owing to its inability to sense Ca²⁺ influx (Littleton et al., 1999). Nevertheless, apart from an involvement in regulating hippocampal brain-derived neurotrophic factor (BDNF) release (Dean et al., 2009), the physiological function of Syt4 remains largely unknown.

In this issue of Neuron, Zhang et al. provide a strong case for the role of Syt4 in the pathogenesis of HFD-induced obesity through the regulation of oxytocin release. In an effort to identify potential regulators for neuropeptide release, Zhang et al. noticed that Syt4 is enriched in adult hypothalamic neurons. Of interest, HFD feeding up-regulates Syt4 expression specifically in the hypothalamus, suggesting a possible role of Syt4 in HFD-induced obesity. To test this possibility, they exposed Syt4 deficient syt4^−/− mice to HFD. On normal chow diet, syt4^−/− mice exhibit a grossly normal phenotype, suggesting a permissive role for Syt4 under normal conditions. However, when challenged by HFD, syt4^−/− mice are completely protected from HFD-induced obesity, due to reduced caloric intake and increased energy expenditure. To ascertain the role of hypothalamic Syt4 in obesity regulation, the authors elegantly used shRNA-medicated knockdown of Syt4 to specifically reduce Syt4 expression in the hypothalamus. Results show that Syt4 knockdown protects mice against HFD-induced obesity. Collectively, these data demonstrate that expression of hypothalamic Syt4 is required for the manifestation of HFD-induced obesity.

To reveal the identity of neurons that mediate Syt4 function, Zhang et al. examined the colocalization of Syt4 with known hypothalamic neuropeptides. Interestingly, Syt4 is exclusively localized with oxytocin, but not found in nearby arginine vasopressin (AVP) neurons. Further electron microscopic studies revealed that Syt4 is expressed in both dense and small vesicles located in axonal terminals and dendrites. The expression in axonal terminals may reflect a role of Syt4 in modulating Ca²⁺ induced vesicle fusion in the posterior pituitary (Zhang et al., 2009). The expression in dendrites is consistent with dendritic release of oxytocin. Thus, it appears that oxytocin neurons mediate the effect of Syt4.

Is Syt4 expression in oxytocin neurons sufficient to render an obesogenic effect? To test this, Zhang et al. used a combination of a viral vector and the oxytocin promoter to over-express Syt4 specifically in oxytocin neurons of wild type and syt4^−/− mice. In both cases, over-expression leads to body weight gain associated with higher food intake, suggesting that the expression of Syt4 in oxytocin neurons is sufficient for the development of obesity. Interestingly, the obesity associated with Syt4 over-expression is abrogated by pharmacological application of oxytocin, suggesting that the obesogenic action of Syt4 is mediated by oxytocin. If this is the case, as reflected by an earlier study showing that Syt4 diminishes BDNF release (Dean et al., 2009), then expression of Syt4 should reduce oxytocin release. Indeed, oxytocin release is increased from syt4^−/− PVH slices relative to wild type mice under both basal and KCl-evoked conditions. Consistently, compared to wild type mice, the serum oxytocin levels are almost doubled on chow diet and tripled on HFD in syt4^−/− mice. This dramatic increase in the oxytocin level is due to specific deletion of Syt4 in oxytocin neurons since this effect is abrogated by the over-expression of Syt4 specifically in these neurons. These results demonstrate that Syt4 profoundly and specifically diminishes oxytocin release, which represents a novel mechanism by which hypothalamic neurons regulate body weight.

Is the increased oxytocin release responsible for the complete protection from HFD-induced obesity in syt4^−/− mice? To investigate this, Zhang et al. blocked the action of oxytocin in syt4^−/− mice by either applying the oxytocin receptor antagonist OVT, or by knocking down oxytocin expression in the PVH. In both cases, the anti-obesity effect of Syt4 deficiency is diminished, thereby suggesting that the augmented oxytocin action is required for resistance to HFD-induced obesity in syt4^−/− mice. This result is corroborated by the effect of oxytocin on preventing HFD-induced obesity presented by the authors and the anorexigenic effect of oxytocin previously reported by others (Blevins et al., 2004; Kublaoui et al., 2008). One prediction based on these results is that mice with oxytocin deficiency will be more sensitive to HFD-induced obesity, which is yet to be tested.

How does HFD up-regulate Syt4 expression in oxytocin neurons? Syt4 expression can be induced by increased neuron activity and intracellular cAMP (Vician et al., 1995). Once might speculate that the Syt4 induction is mediated by the melanocortin receptor 4 (MC4R), a critical regulator for body weight homeostasis. This speculation is supported by the increased MC4R that has been observed in the PVH upon HFD feeding (Enriori et al., 2007), which is thought to be coupled with increased cAMP. Is oxytocin the only mediator for the resistance to HFD-induced obesity in syt4^−/− mice? Both pharmacological blockage of oxytocin action and knockdown of oxytocin expression in syt4^−/− mice only partially reversed the anti-obesity effect by Syt4 deficiency. This result may indicate a role for additional neurotransmitters from oxytocin neurons although technical limitations could also underlie the partial effects. Considering the association of Syt4 with small vesicles, the release of neurotransmitters contained in small vesicles will also be similarly affected by Syt4 deficiency. In this regard, oxytocin neurons are glutamatergic and whether glutamate release mediates anti-obesity effect by Syt4 deficiency should also be an interesting future study. In addition, according to the Alan Brain Atlas gene expression database, Syt4 is expressed in a subset of hindbrain neurons, a brain site also heavily involved in feeding and body weight regulation. Whether Syt4 is similarly induced in these areas by HFD and whether these neurons contribute to the anti-obesity function of Syt4 deficiency await further studies. Mechanistically, it remains to be established how increased Syt4 leads to reduced oxytocin release. Notably, it appears that the dramatic increase in oxytocin release by Syt4 deficiency can’t entirely be explained by the previous observation that in syt4^−/− mice, low Ca2+ entry triggers more release while high Ca2+ entry triggers less release from axon terminals of the posterior pituitary (Zhang et al., 2009). These axon terminals are presumably from oxytocin neurons since only oxytocin and AVP neurons send projections to the posterior pituitary and Syt4 is not expressed in AVP neurons. The reason for this discrepancy is unknown but may involve different approaches used in the two studies (electrophysioligical recordings versus peptide release assay). In light of Syt4 localization in vesicles, the ability of Syt4 to form the fusion complex, and the inability to sense Ca²⁺, it can be hypothesized that the increased Syt4 prevents Ca²⁺ -mediated vesicle exocytosis, as previously demonstrated (Littleton et al., 1999).

Can Syt4 be an effective anti-obesity drug target? Although more proof of concept studies are required before targeting Syt4 for obesity treatment, the following functional characteristics of Syt4 provided by Zhang et al. argue that it could be an attractive drug target for the current obesity epidemic: 1) permissive role of Syt4 at the basal level (i.e. on chow diet); 2) abundant Syt4 only found in a few brain sites in adult mice; 3) specific induction of Syt4 in oxytocin neurons by HFD and 4) robust effects of Syt4 deficiency on the prevention of HFD-induced obesity. These characteristics should in principle form a basis for developing effective anti-obesity drugs.

In sum, the study by Zhang et al. nicely bridges the gap in our understanding of the physiological function of Syt4 and the mechanism of HFD-induced obesity. The authors employed an elegant combination of mouse genetics, RNA interference, stereotaxic injection and viral directed expression approaches, and have provided comprehensive evidence for an obesogenic oxytocin neuron-specific program. This program includes HFD-induced Syt4 expression, reduced oxytocin release, increased caloric intake and reduced energy expenditure, and ultimately the manifestation of obesity. This program represents a previously unknown mechanism for obesity pathogenesis. Do other neuropeptide-releasing neurons contribute to this novel mechanism? More investigations stemming from the findings by Zhang et al. will answer this question in the years to come.