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C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro
1Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Insights
The C-peptide of human preproinsulin-like peptide 7 (C-INSL7) is present in rat brain neurons and influences neuronal activity. C-INSL7 acts independently from insulin-like peptide 7 (INSL7), suggesting a distinct signaling role in the central nervous system.
Area of Science:
- Neuroscience
- Endocrinology
- Molecular Biology
Background:
- The central nervous system (CNS) utilizes various signaling molecules to regulate neuronal function.
- Insulin-like peptide 7 (INSL7) and its related peptides are implicated in diverse physiological processes.
- The specific role and distribution of C-peptide of human preproinsulin-like peptide 7 (C-INSL7) within the CNS remain largely unexplored.
Purpose of the Study:
- To investigate the presence and distribution of C-INSL7 immunoreactivity in the rat brain.
- To examine the functional effects of C-INSL7 on cultured rat hypothalamic neurons.
- To determine if C-INSL7 and INSL7 bind to distinct receptors in the rat brain.
Main Methods:
- Immunohistochemistry was employed to detect C-INSL7-immunoreactive neurons and fibers in rat brain sections.
- Optical imaging using voltage-sensitive dye DiSBAC4(3) and calcium-sensitive dye fura-2 was used to assess neuronal responses to C-INSL7 and INSL7.
- Radioligand binding assays with [125I]C-INSL7 were performed to characterize binding sites in rat brain membranes.
Main Results:
- C-INSL7-immunoreactive neurons were identified in specific brain regions, including the pontine nucleus incertus and hypothalamus, with extensive fiber distribution.
- C-INSL7 induced dose-dependent changes in neuronal membrane potential and intracellular calcium levels in cultured hypothalamic neurons.
- Binding studies revealed that C-INSL7 binds to distinct sites in rat brain membranes, separate from INSL7 binding sites.
Conclusions:
- C-INSL7 is expressed in specific neuronal populations within the rat CNS.
- C-INSL7 exerts direct functional effects on hypothalamic neurons, modulating their electrical activity and calcium signaling.
- These findings suggest that C-INSL7 functions as an independent signaling molecule within the rat central nervous system.
Abstract:
With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.
