In recent years, nesfatin-1 [NESF-1; processed from nucleobindin-2/NUCB2] has received much attention due to its roles in metabolism, stress, and anxiety. NESF-1 and nesfatin-1-like-peptide (NLP) affect synthesis of stress hormones. The release of glucocorticoids [GC] as end products in the stress/HPA axis modulates metabolism, potentially impacting energy availability through catabolic processes. In animal models, corticosterone was shown to increase the intake of palatable foods, including carbohydrates and lard. These findings might explain how repeated stress-related GC secretion leads to the intake of high-calorie food and weight gain. Animals vulnerable to obesity had higher circulating GC, and GC antagonists prevented/reversed the weight gain in these animals. Some human studies reported that abdominal obesity might be associated with elevated GCs in response to stress. Food intake was significantly increased immediately after stress, showing that this response was related to the stress reactivity but not the total secreted cortisol. Furthermore, the infusion of corticotropin-releasing hormone [CRH] at physiological doses significantly increased food intake in humans compared to placebo-injected non-obese adults. CRH initiates the HPA axis. Various neurons expressing NUCB2/NESF-1 are colocalized with CRH in hypothalamic paraventricular nucleus. NUCB2/NESF-1 increased the excitability of CRH neurons. NESF-1 was shown to enhance cytoplasmic Ca2+ levels in CRH neurons. Moreover, bilateral adrenalectomy increased NESF-1 mRNA in the rat hypothalamus. Less than 50% of neurons expressing NESF-1 in PVN and arcuate nucleus contain glucocorticoid receptors. These findings suggests that central NESF-1 could affect CRH neurons in PVN and initiate central and peripheral HPA axis responses. Two reports showed that NESF-1 might apply its anorectic effect through CRH and its receptor [CRHR]-mediated system. In both studies, intracerebroventricular [ICV] injection of NESF-1 decreased food intake, in rats during the dark phase, and in neonatal chicks. However, CRHR antagonists abolished the anorectic effect of NESF-1.
Very recently, it has been proposed that a NUCB2-related peptide, nucleobindin-1 [NUCB1], possesses a nesfatin-1-like peptide (NLP) sequence, which could be processed by prohormone convertases. NUCB1 and NUCB2 shared 60% sequence homology in the mouse genome. It was reported that the bioactive core of NLP and NESF-1 shared 76% amino acid sequence homology in mouse. The presence of NLP was reported in tissues in which NUCB2/NESF-1 was previously identified, including pancreas, pituitary, gonads, and gut. Similarly, NLP was shown to suppress food intake and modulate the expression of appetite-regulatory hormones in goldfish and rats.
Previous studies from our lab also showed that NESF-1 is a stress-responsive peptide that stimulates stress-related hypothalamus-pituitary-interrenal [HPI; similar to mammalian HPA tissues] axis hormones in goldfish. Moreover, NESF-1 was shown to directly stimulate the synthesis of ACTH precursor in mouse pituitary corticotrophs. NESF-1 is a catabolic hormone with satiety effects and stimulates ACTH precursor, which is the primary pituitary regulator of another catabolic hormone, cortisol. Moreover, NESF-1 binding sites were detected in the adrenal gland of rats. Based on this evidence, especially the positive roles of NESF-1 or NLP on ACTH, we hypothesized that they elicit a similar stimulatory role on cortisol synthesis and secretion. The objective of this study was to assess whether adrenal cortex cells express NUCB1/NLP and NUCB2/NESF-1 and whether they directly act on adrenal cortical cells to modulate the synthesis of cortisol and modulate steroidogenic enzymes involved in this pathway in vitro and in vivo.
Human adrenal cortex (H295R) cells were immunoreactive for both NUCB1/NLP and NUCB2/NESF-1. Both NUCB1/NLP-like and NUCB2/NESF-1-like immunoreactivity showed a diffuse distribution in both cytoplasm [Green] and nucleus [DAPI; blue] in Fig.1A,B. No immunoreactivity was observed in control groups that were only incubated with the secondary antibodies (Fig.1C).
The cortisol content of H295R cells at different time points [6–24 h] is shown in Fig.2. NLP and NESF-1 at 10 nM decreased cortisol content of H295R cells at 24 h post-incubation, while no such effects were found at other time points tested. Meanwhile, ACTH [100 nM; positive control] enhanced cellular cortisol content at 6, 12 and 24 h post-incubation.
As shown in Fig.3A, NLP and NESF-1 decreased P-CREB/T-CREB ratio by more than twofold compared to the control group. Forskolin enhanced cortisol secretion significantly when compared to the no-treatment control group. When H295R cells were preincubated with either NESF-1 or NLP, the stimulatory effects of forskolin, the classical activator of adenylyl cyclase, on cortisol release was significantly lower compared to the group incubated with forskolin alone (Fig.3B).
