Minireview: Nesfatin-1—An Emerging New Player in the Brain-Gut, Endocrine, and Metabolic Axis

Abstract

Nesfatin-1 is a recently identified 82-amino-acid peptide derived from the precursor protein, nucleobindin2 (NUCB2). The brain distribution of NUCB2/nesfatin-1 at the mRNA and protein level along with functional studies in rodents support a role for NUCB2/nesfatin-1 as a novel satiety molecule acting through leptin-independent mechanisms. In addition, nesfatin-1 induces a wide spectrum of central actions to stimulate the pituitary-adrenal axis and sympathetic nervous system and influences visceral functions and emotion. These central actions combined with the activation of NUCB2/nesfatin-1 neurons in the brain by various stressors are indicative of a role in the adaptive response under stressful conditions. In the periphery, evidence is mounting that nesfatin-1 exerts a direct glucose-dependent insulinotropic action on β-cells of the pancreatic islets. However, the cellular mechanisms of nesfatin-1's action remain poorly understood, partly because the receptor through which nesfatin-1 exerts its pleiotropic actions is yet to be identified.

Nesfatin-1, an 82-amino-acid peptide identified by Oh-I et al. (1), was named after its first described biological action to curtail the dark-phase food intake and weight gain in rats [nucleobindin2 (NUCB2)-encoded satiety- and fat-influencing protein]. The 396-amino-acid precursor protein NUCB2 is highly conserved among rodents and humans as well as within nonmammalian vertebrate species indicative of its importance (2). Proteolytic processing of NUCB2 by prohormone convertases may result, in addition to the N-terminal fragment, nesfatin-1 (residues 1–82), in two C-terminal peptides, nesfatin-2 (residues 85–163) and nesfatin-3 (residues 166–396), although only nesfatin-1 has the ability to reduce the dark-phase food intake (1, 3).

Localization of NUCB2/Nesfatin-1 in the Brain and Periphery

NUCB2 expression at the gene and protein levels was originally described in rat hypothalamic nuclei involved in feeding regulation, namely the paraventricular nucleus (PVN), supraoptic nucleus (SON), lateral hypothalamic area, and the arcuate nucleus (1). This initial brain mapping was subsequently extended to include the localization of NUCB2/nesfatin-1-immunoreactive cells in cortical, limbic, pontine, and medullary nuclei involved in stress- and autonomic-related responses such as the insular cortex, central amygdaloid nucleus, dorsomedial hypothalamic nucleus, tuberal hypothalamic area, periventricular nucleus, Edinger-Westphal nucleus, locus coeruleus, the medullary raphe nuclei, ventrolateral medulla, cerebellum, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and preganglionic sympathetic and parasympathetic neurons of the spinal cord in rats (4–9) and mice (10). Another step forward was the biochemical characterization of hypothalamic cells coexpressing NUCB2/nesfatin-1 immunoreactivity with other peptides involved in the regulation of food intake [melanin-concentrating hormone, cocaine- and amphetamine-regulated transcript, α-MSH, proopiomelanocortin, and neuropeptide Y (NPY)], pituitary hormone secretion [vasopressin, oxytocin, GHRH, CRH, corticotropin-releasing factor (CRF), TRH, and somatostatin], and stress response (CRF, neurotensin, urocortin, and serotonin) (4–8, 11, 12). It is worth noting that all the brain-mapping studies have been performed with antibodies specific to nesfatin-1 but also recognizing full-length NUCB2. Therefore, the expression pattern cannot distinguish whether the distribution reflects the precursor and/or the processed nesfatin-1. At the cellular level, a consistent feature of NUCB2/nesfatin-1 immunostaining is the confinement to cell body cytoplasm and primary dendrites, whereas immunolabeling is completely absent from axons and nerve terminals (1, 4, 5, 7, 9, 10). Such cellular localization is at variance with other neuropeptides and points toward an intracellular rather than extracellular mode of action of central NUCB2/nesfatin-1. However, future investigations are needed to delineate the subcellular localization of NUCB2/nesfatin-1 at the electron microscopic level and to clarify whether there is a postsecretory processing of NUCB2 to nesfatin-1.

In addition to the wide distribution of NUCB2/nesfatin-1 in the central nervous system, NUCB2 mRNA is expressed in the periphery including the pituitary, stomach, pancreas, testis, and adipose tissue in rodents and goldfish (2, 13–16). Of relevance, we found 10-fold higher levels of NUCB2 mRNA detected by quantitative RT-PCR in the rat gastric oxyntic mucosa compared with the brain (13). At the cellular level, NUCB2/nesfatin-1 immunostaining was detected solely in the cytoplasm of endocrine cells, namely the ghrelin-containing X/A-like cells and, to a much smaller extent, somatostatin and enterochromaffin-like cells of the gastric corpus oxyntic mucosa, β-cells of the pancreas, and cells of the pituitary gland anterior lobe in rats and mice (5, 13, 16). Confocal microscopy of single X/A-like cells indicated that NUCB2/nesfatin-1 is localized in a different pool of cytoplasmic vesicles from ghrelin (13). Likewise, in pancreatic β-cells, the subcellular distribution of NUCB2/nesfatin-1 and insulin is not identical (15). These data, along with the distinct response of circulating NUCB2/nesfatin-1 to a meal compared with that of insulin or acyl ghrelin in healthy subjects (17) support a differential regulation and release of these hormones. However, the prominent labeling of NUCB2/nesfatin-1 in endocrine cells and adipose tissue strongly suggests a role as a circulating hormone involved in various homeostatic processes. This is further supported by studies in mice showing that circulating nesfatin-1, like other gut hormones influencing food intake (18), can cross the blood-brain barrier bidirectionally by a nonsaturable mechanism (19, 20). A recent clinical study provided indirect evidence that a similar process may occur in humans as shown by the significant linear relation between plasma NUCB2/nesfatin-1 and cerebrospinal fluid NUCB2/nesfatin-1 levels in both lean and, to lesser degree, in fasted obese subjects (21).

A relevant aspect still to be clarified is whether NUCB2 processing yields the mature nesfatin-1_1–82. Until now, outside the original report describing the presence of the 10-kDa peptide in the cerebrospinal fluid of rats (1), none of the subsequent studies in rat or goldfish brain tissue (2, 5, 22), rat gastric mucosa, pancreas, pituitary, and adipose tissue (13, 14) were able to show fully processed mature nesfatin-1 by Western blot and detected only the full-length NUCB2 protein, whereas synthetic nesfatin-1 as positive control was detectable at the predicted molecular weight (13, 22). This may be indicative of postsecretory processing of NUCB2 to nesfatin-1 particularly because a new sensitive sandwich-type ELISA using N-terminal and C-terminal antibodies without cross-reaction with full-length NUCB2 or nesfatin-2 and nesfatin-3 detected nesfatin-1 in human plasma samples (17).

Nesfatin-1 as a New Anorexigenic Hormone

The first biological action ascribed to NUCB2 and nesfatin-1 was the reduction of dark-phase food intake after injection into the third brain ventricle in rats and a concomitant reduction in body weight gain and fat pads upon subchronic administration (1). Now, compelling functional tests showed that nesfatin-1 injected into the brain elicits an anorexigenic response in rodents (3, 11, 23–26) as well as in goldfish (2). Recent automated microstructure analysis of meal patterns in mice documented that the anorexigenic effect of nesfatin-1 is due to the induction of satiation (reduction of meal size) as well as satiety (decreased meal frequency associated with prolonged intermeal intervals) during the first 4 h after lateral brain ventricle injection in the dark phase (23). In addition to its action in the forebrain within the PVN (11), nesfatin-1 injected into the cisterna magna or the fourth ventricle also reduced the dark-phase food intake within the first hour after injection (24) indicative of specific hindbrain sites of action yet to be localized by microinjection studies.

Because most peptides in the brain that influence food intake also affect digestive processes (27), a central modulation of gastrointestinal propulsive motor function was also assumed for nesfatin-1. It was demonstrated that nesfatin-1 injected into the lateral brain ventricle delayed gastric emptying in rats (24) as well as in mice (10) and suppressed gastroduodenal motility in mice (26), an effect that could contribute to the induction of satiety. In the brain, the underlying mechanisms of nesfatin-1's anorexigenic action have been reproducibly established to be independent of leptin (1, 11, 28) although involving several other hypothalamic and medullary pathways regulating food intake. Those include the activation of the anorexigenic CRF receptor 2 (CRF₂) signaling system (29, 30) because the injection of CRF₂ antagonist, astressin₂-B into the lateral brain ventricle blocked the decrease of food intake in response to nesfatin-1 injected by the same route (24). Interestingly, astressin₂-B did not modify the reduction of food intake when nesfatin-1 was injected into the brain medulla at the level of the cisterna magna (24), further supporting differential forebrain and hindbrain downstream signaling cascades of nesfatin-1. Additionally, the melanocortin 3/4 receptor antagonist SHU9119 (1, 31) as well as an oxytocin antagonist (11, 25) blocked the forebrain nesfatin-1-induced reduction of food intake in rats. Whether nesfatin-1 acts either in series or in parallel to recruit CRF₂, melanocortin 3/4 and oxytocin signaling pathways is still to be clarified. In addition, nesfatin-1 may also suppress orexigenic pathways such as NPY signaling based on an in vitro study using whole-cell current clamp recordings from rat arcuate neurons showing a hyperpolarization of NPY-positive neurons when nesfatin-1 was applied to the cell (32). Moreover, because nesfatin-1 selectively reduces dark-phase food intake in ad libitum-fed animals (1, 11, 24, 31), whereas during the light phase, inconsistent results were observed in the feeding response to a fast (24, 31), these differential actions may be indicative of a specific interaction with orexigenic mechanisms physiologically recruited during the dark phase compared with the hyperphagic response to a fast (33–35) that needs to be substantiated in future investigations.

Convergent sets of evidence support a physiological role for NUCB2/nesfatin-1 as a negative modulator of food intake. First, the most abundant mRNA/protein expression of NUCB2/nesfatin-1 is detected in hypothalamic nuclei and brainstem areas established to play a pivotal role in regulating food intake and metabolism (5, 7, 9–11). Functional studies established that nesfatin-1 or NUCB2 injected into the cerebrospinal fluid at picomolar doses induce a reproducible anorexigenic effect in rodents and goldfish (1, 2, 24, 25). Second, the hypothalamic mRNA and protein expression of NUCB2 in the PVN and SON varies with alterations of energy status being down-regulated after short or chronic undernutrition and conversely up-regulated after refeeding in rats (1, 7, 22) and goldfish (2). In addition, other food intake-suppressing transmitters such as α-MSH or cholecystokinin increase NUCB2 mRNA expression (1) or activate NUCB2/nesfatin-1-immunopositive neurons in hypothalamic and brainstem nuclei (24, 36). Lastly, injection of an anti-NUCB2 antisense oligonucleotide or anti-nesfatin-1 antibody into the third brain ventricle increases food intake in male rats (1). However, a recent study showed that the effective knocking-down of hypothalamic NUCB2 had no effect on food intake or body weight gain in female rats (22). Whether these disparate results reflect a sex difference needs to be clarified. In addition, the cellular mechanisms of nesfatin-1's anorexigenic action remains ill defined due to the current lack of identification of the receptor. One report suggests an interaction with a G protein-coupled receptor leading to an increase of Ca²⁺ concentration linked with protein kinase A in cultured cells isolated from the rat hypothalamus (4).

In contrast to the majority of studies investigating the effects of centrally injected nesfatin-1 on food intake, reports on the influence of peripherally administered nesfatin-1 on food intake are more limited, and results are less consistent. One group of investigators showed that nesfatin-1 as well as nesfatin-1_24–53 (30-amino-acid midsegment) reduces the dark-phase food intake after ip injection of a large dose in ad libitum-fed mice through leptin-independent mechanisms (37). The peripheral effects are likely mediated via the vagus nerve based on in vitro data showing that nesfatin-1 activates Ca²⁺ influx in primary cultured nodose ganglion neurons from mice (38) and the finding that mice pretreated with capsaicin do not respond with a reduction of food intake to nesfatin-1_24–53 injected ip (39). However, in other reports, ip injection of nesfatin-1 at a similarly high dose did not alter dark-phase food intake in a different strain of mice (23) as well as in rats (24). In goldfish, no decrease or an 18% decrease of food intake was observed at a dose that was 10^2–3-fold higher than that injected intracerebroventricularly, leading to a striking food intake reduction (2). Therefore, the satiety action of nesfatin-1 is more readily induced by central than peripheral injection. This warrants further investigation particularly in the context of high gastric NUCB2 mRNA expression and circulating levels of NUCB2/nesfatin-1 being regulated by changes in nutritional status as shown by the significant 18% reduction of plasma levels after 24 h fasting and the return to baseline after refeeding in rats (13). Moreover, a recent clinical study in healthy nonobese male subjects showed a negative correlation between body mass index and fasting plasma levels of nesfatin-1 measured using a sensitive nesfatin-1-specific ELISA, consistent with the possibility that nesfatin-1 may play a role in body weight regulation in humans (17). However, the nutrient-related fluctuation of nesfatin-1 plasma levels is not occurring in healthy humans in response to a meal under conditions where acyl ghrelin decreases or insulin increases (17, 40). Other studies in patients described a positive correlation between NUCB2/nesfatin-1 plasma levels and body mass index with lower levels under conditions of anorexia nervosa (41) and higher levels in obese subjects (14, 21) indicative of circulating nesfatin-1 levels being possibly regulated by sustained changes in adipose tissue mass. The recently described lower ratio of cerebrospinal fluid/plasma NUCB2/nesfatin-1 in obese compared with lean subjects may suggest a reduced uptake from the circulation to the brain and therefore a reduced central action to influence food intake/body weight (21). Moreover, the reported difference in eight single-nucleotide polymorphisms in the NUCB2 gene associated with obesity in a large cohort of subjects may provide a genetic base for the susceptibility to or protection against the development of obesity (42).

Involvement of Nesfatin-1 in Neuroendocrine and Stress Responses

The hormone CRF is the hallmark initiator of the stress response (43). Various stressors stimulate the release of CRF, which consecutively stimulates the hypothalamic-pituitary-adrenal (HPA) axis, modulates behavior, activates the sympathetic nervous system, and alters visceral functions (43–45). Converging neuroanatomical and functional studies support a role of nesfatin-1 as part of neuropeptides being activated and potentially playing a role as effector of stress response. This is based on the colocalization of NUCB2/nesfatin-1 with CRF in the PVN (4) with 24% of all nesfatin-1-immunoreactive cells also expressing CRF (5). Endocrine studies recently showed that third ventricular injection of nesfatin-1 activates the HPA axis assessed by the elevated plasma ACTH and corticosterone levels, likely mediated via an activation of CRF neurons in the PVN (46). Other functional studies demonstrate a role of brain CRF₂ receptors in the inhibition of food intake induced by intracerebroventricularly injected nesfatin-1 (5 pmol) (24). There is also mimicry between nesfatin-1 and CRF injected centrally to induce similar behavioral responses encompassing anxiety and fear (43, 47), sympathetic activation (45, 48), hypertension (25, 44), and delayed gastric emptying (10, 24, 26, 49). Moreover, convergent reports show that various stressors such as abdominal surgery (50), injection of lipopolysaccharide (51), and restraint stress (12, 46, 52–54) result in the activation of nesfatin-1-immunoreactive neurons in stress-responsive circuitries namely in the anterior parvicellular part of the PVN, the Edinger-Westphal nucleus and nuclei of the catecholaminergic and serotonergic systems as well as hindbrain nuclei. However, after restraint stress, plasma nesfatin-1 levels were not altered (46), pointing toward a purely central mode of action.

Nesfatin-1 as a Novel Glucose-Dependent Stimulant of Insulin

Nesfatin-1 immunoreactivity has been described in the human and rodent pancreas and is located exclusively in β-cells containing insulin (15, 16, 55), suggesting a role in the control of glucose/insulin regulation. Existing evidence indicates that nesfatin-1 stimulates glucose-induced insulin release and preproinsulin mRNA expression in rat or mouse isolated islets or cultured MIN cells (55, 56) by promoting calcium influx through L-type channels (56). Such a direct glucose-dependent insulinotropic effect of nesfatin-1 on β-cells is further supported by the in vivo demonstration that iv injection of nesfatin-1 reduces blood glucose levels in hyperglycemic db/db mice and in nonhyperglycemic mice, whereas central injection does not (57). In Goto-Kakizaki rats with type 2 diabetes, the level of NUCB2 immunoreactivity in islet homogenate is reduced compared with Wistar rats and plasma NUCB2 concentrations showed an inverse relationship with circulating glucose levels during the glucose tolerance test (15). This negative association between blood glucose and nesfatin-1 in Goto-Kakizaki rats was also observed in human subjects with type 2 diabetes (40). Similarly, nesfatin-1 levels were lower in breast milk of human subjects with gestational diabetes (58). Altogether, these data support an alteration of NUCB2/nesfatin-1 expression in type 2 diabetes, which may have a bearing on the impaired glucose homeostasis. By contrast, in healthy subjects, an oral glucose tolerance test did not modify circulating nesfatin-1 levels (17, 40), which is, however, consistent with experimental studies pointing to a primarily local action of NUCB2/nesfatin-1 in the regulation of the endocrine pancreas (15).

Conclusions

Central nesfatin-1 has recently emerged as physiological negative regulator of food intake based on its expression in key food intake-regulatory nuclei in the hypothalamus and brainstem, the regulation of hypothalamic NUCB2/nesfatin-1 mRNA and protein expression upon changes in nutritional status, and the increased food intake after neutralization of central endogenous NUCB2/ nesfatin-1 in male rats. The food intake-suppressing effect is independent of leptin and associated with the stimulation of brain CRF₂, melanocortin_3/4, and oxytocin receptor signaling systems. In addition, NUCB2/nesfatin-1 neurons in the brain are activated by various stressors, and stress/CRF-like effects are induced by central injection of nesfatin-1, namely the stimulation of the HPA axis and sympathetic activity, anxiogenic behavior, and alterations of visceral functions (rise in blood pressure and suppression of gastric emptying). A recent study also provides evidence for a key role of nesfatin-1 in the development of normal puberty onset in female rats (22). Importantly, the mounting evidence that NUCB2/nesfatin-1 located in pancreatic β-cells acts directly in these cells as a glucose-dependent insulinotropic peptide may open new venues for the treatment of type 2 diabetes. Collectively, studies available so far support an expanding role of nesfatin-1, opening new fields of investigation. However, many questions are still to be answered, in particular the receptor involved in the peptide's actions, and the processing of NUCB2 to nesfatin-1 in hypothalamic or gut tissues still remains elusive. Lastly, signaling mechanisms directly associated with the action of nesfatin-1 have been little explored.