Identification and Characterization of Nesfatin-1 Immunoreactivity in Endocrine Cell Types of the Rat Gastric Oxyntic Mucosa

Abstract

Hypothalamic nesfatin-1, derived from the nucleobindin2 (NUCB2) precursor, inhibits nocturnal food intake and body weight gain in rats. Nesfatin-1 is able to cross the blood-brain barrier, suggesting a peripheral source of nesfatin-1. Many centrally acting food intake regulatory neuropeptides are also produced in the periphery, especially in the gastrointestinal tract. Therefore, we investigated the gene expression of NUCB2 and distribution of nesfatin-1-immunoreactive cells in the stomach. Microarray mRNA expression profiles in purified small endocrine cells of the gastric mucosa substantiated by quantitative RT-PCR showed significantly higher NUCB2 mRNA expression compared with brain and heart. Western blot confirmed the expression of NUCB2 protein and its transport into a secretory soluble fraction of gastric mucosal endocrine cell homogenates. Immunohistochemical colabeling for nesfatin-1 and ghrelin, histidine decarboxylase, or somatostatin revealed two subtypes of nesfatin-1-positive endocrine cells. Cells in the midportion of the glands coexpressed nesfatin-1 and ghrelin, whereas few cells in the glandular base coexpressed nesfatin-1 and somatostatin or histidine decarboxylase. High-resolution three-dimensional volume imaging revealed two separate populations of intracytoplasmic vesicles in these cells, one containing nesfatin-1 and the other ghrelin immunoreactivity. Microarray rat genome expression data of NUCB2 in small gastric endocrine cells confirmed by quantitative RT-PCR showed significant down-regulation of NUCB2 after 24 h fasting. In summary, NUCB2 mRNA expression as well as protein content is present in a specific subset of gastric endocrine cells, most of which coexpress ghrelin. NUCB2 gene expression is significantly regulated by nutritional status, suggesting a regulatory role of peripheral nesfatin-1 in energy homeostasis.

Lipopolysaccharide Increases Gastric and Circulating NUCB2/Nesfatin-1 Concentrations in Rats

“…In the present study, only the full length NUBC2 was detected by Western blot. The antibody used was raised against mature nesfatin-1 amino acids 1-82 and shown before to detect synthetic nesfatin-1 peptide as well as full length NUCB2 which also contains the epitope [36]. So far, only one study described the occurrence of mature nesfatin-1 peptide in the cerebrospinal fluid of rats, whereas in brain nuclei only full length NUCB2 was detected [24].…”

“…So far, only one study described the occurrence of mature nesfatin-1 peptide in the cerebrospinal fluid of rats, whereas in brain nuclei only full length NUCB2 was detected [24]. In all subsequent reports, only full length NUCB2 was detected by Western blot [8,27,36] or studies performing immunostaining did not distinguish between NUCB2 protein and nesfatin-1 peptide [5,6,8,10,13,14,16,18,25,32,34,42,43]. Since also full length NUCB2 exerts an anorexigenic effect it is also possible that this protein is the active circulating form.…”

“…However, we recently identified the expression of another product in these cells, nucleobindin2 (NUCB2) [36] which may be cleaved in the hypothalamus into nesfatin-1, nesfatin-2 and nesfatin-3 [24]. NUCB2/ nesfatin-1 was described in rat brain food intake regulatory nuclei and implicated in the regulation of food intake [24].…”

Effect of fundectomy, antrectomy and gastrectomy on pancreatic and brush border enzyme activity in rats

“…Nesfatin-1 was detected mainly within the central nervous system, in the arcuate nucleus, supraoptic nucleus, hypothalamus, and spinal cord (13). It was contained in the central part of the pancreatic islets, liver, spleen, thymus, heart, liver, muscle, pituitary glands, testes, fat tissue, and endocrine cells of the digestive tract (13,29,33). As an anorectic hormone, nesfatin-1 takes part in appetite regulatory processes, in regulation of body weight, fat mass, glucose levels, insulin secretion, and energy homeostasis (14,29).…”

“…It was contained in the central part of the pancreatic islets, liver, spleen, thymus, heart, liver, muscle, pituitary glands, testes, fat tissue, and endocrine cells of the digestive tract (13,29,33). As an anorectic hormone, nesfatin-1 takes part in appetite regulatory processes, in regulation of body weight, fat mass, glucose levels, insulin secretion, and energy homeostasis (14,29). Although it has been found that nesfatin-1 influences gastrointestinal motility and reduces gastric acid secretion (2,30,31), the functional roles of this peptide in the digestive tract are not completely understood.…”