Roberta Possenti

The Extended Granin Family: Structure, Function, and Biomedical Implications

The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers.

VGF: A Novel Role for This Neuronal and Neuroendocrine Polypeptide in the Regulation of Energy Balance ☆

Insight into the mechanisms of action of neurotrophic growth factors has been obtained through the identification and characterization of gene products that are regulated or modified at the transcriptional, translational, and/or posttranslational level in response to neurotrophin treatment. VGF (non-acronymic) was identified approximately 15 years ago as a nerve growth factor (NGF)-regulated transcript in rat PC12 pheochromocytoma cells. Subsequent studies have demonstrated that neurotrophins such as NGF and brain-derived neurotrophic factor induce vgf gene expression relatively rapidly in PC12 cells and cultured cortical neurons, respectively, in comparison to less robust regulation by epidermal growth factor (EGF) and insulin, growth factors which do not trigger the neuronal differentiation of PC12 cells. vgf gene expression is stimulated in vitro by NGF and the ras/map kinase signaling cascade through a CREB-dependent mechanism, while in vivo, VGF mRNA levels are regulated by neuronal activity, including long-term potentiation, seizure, and injury. Both the mRNA and encoded approximately 68-kDa protein (VGF) are selectively synthesized in neuroendocrine and neuronal cells. The predicted VGF sequence is rich in paired basic amino acid residues that are potential sites for proteolytic processing, and VGF undergoes regulated release from dense core secretory vesicles. Although VGF mRNA is synthesized widely, by neurons in the brain, spinal cord, and peripheral nervous system, its expression is particularly abundant in the hypothalamus. In addition, VGF peptides are found in hypophysial, adrenal medullary, gastrointestinal, and pancreatic endocrine cells, suggesting important neuroendocrine functions. Recent analysis of VGF knockout mice indeed demonstrates that VGF plays a critical role in the control of energy homeostasis. VGF knockout mice are thin, small, hypermetabolic, hyperactive, and relatively infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic pro-opiomelanocortin, neuropeptide Y, and agouti-related peptide expression. Coupled with the demonstration that VGF mRNA levels are induced in the normal mouse hypothalamic arcuate nuclei in response to fasting, important central and peripheral roles for VGF in the regulation of metabolism are suggested. Here we review previous studies of VGF in the broader context of its newly recognized role in the control of energy balance and propose several models and experimental approaches that may better define the mechanisms of action of VGF.

Processing, Distribution, and Function of VGF, a Neuronal and Endocrine Peptide Precursor

Abstract1. The vgf gene encodes a neuropeptide precursor with a restricted pattern of expression that is limited to a subset of neurons in the central and peripheral nervous systems and to specific populations of endocrine cells in the adenohypophysis, adrenal medulla, gastrointestinal tract, and pancreas. In responsive neurons, vgf transcription is upregulated by neurotrophins, the basis for the original identification of VGF as nerve growth factor- (NGF) inducible in PC12 cells (A. Levi, J. D. Eldridge, and B. M. Paterson, Science229:393–395, 1985).2. In this review, we shall summarize data concerning the transcriptional regulation of vgf in vitro, the structural organization of the vgf promoter as well as the transcription factors which regulate its activity.3. On the basis of in situ hybridization and immunohistochemical studies, the in vivo tissue-specific expression of VGF during differentiation and in the adult will be summarized.4. Parallel biochemical data will be reviewed, addressing the proteolytical processing of the pro-VGF precursor within the secretory compartment of neuroendocrine cells.5. Finally, analysis of the phenotype of VGF knockout mice will be discussed, implying a nonredundant role of VGF products in the regulation of energy storage and expenditure.

TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography–tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 μg/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue β2-AR (β2 adrenergic receptor) and white adipose tissue (WAT) PPAR-δ (peroxisome proliferator-activated receptor δ), β3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor.

The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF‐processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C‐terminal rat VGF nonapeptide, thus indicating that they included the C‐terminus of proVGF. Ectopic expression of the neuroendocrine‐specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site‐directed mutagenesis was used to identify the KRKRKK488 motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR555 site. By the combined use of high‐resolution separation techniques, matrix‐assisted laser desorption/ionization time of flight (MALDI‐ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C‐terminal region of proVGF.

Tissue‐Specific Processing of the Neuroendocrine Protein VGF

Abstract: VGF is a neuroendocrine‐specific gene product that is up‐regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N‐terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C‐terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low‐molecular‐weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.

A novel neuroendocrine gene product: selective VGF8a gene expression and immuno-localisation of the VGF protein in endocrine and neuronal populations

The VGF8a gene was recognised on the basis of its inducibility by NGF in rat pheochromocytoma (PC12) cells. Using immunocytochemistry, we have localised the corresponding VGF protein product in various neuronal groups, including primary sensory and enteric neurons, and in endocrine cells of the adrenal medulla, adenohypophysis and gut. VGF8a gene expression, as detected by RNAse protection analysis, largely correlated with such distribution.

Characterization of a novel peripheral pro-lipolytic mechanism in mice: role of VGF-derived peptide TLQP-21.

The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/β-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.

TLQP‐21, a neuroendocrine VGF‐derived peptide, prevents cerebellar granule cells death induced by serum and potassium deprivation

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP‐21, a VGF‐derived peptide, to protect, in a dose‐ and time‐dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation‐induced cell death. We demonstrated that TLQP‐21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP‐21 significantly activated extracellular signal‐regulated kinase 1/2, serine/threonine protein kinase, and c‐jun N‐terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP‐21 induced significant increase in intracellular calcium (as measured by fura‐2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP‐21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.

Pro-VGF-derived peptides induce penile erection in male rats: possible involvement of oxytocin

The effect of five peptides derived from the C‐terminal portion of rat pro‐VGF (VGF577‐617, VGF588‐617, VGF599‐617, VGF556‐576 and VGF588‐597) on penile erection was studied after injection into the hypothalamic paraventricular nucleus of male rats. VGF577‐617, VGF588‐617, VGF599‐617 and, to a lower extent, VGF588‐597 (0.1–2 µg) induced penile erection episodes in a dose‐dependent manner when injected into the paraventricular nucleus, while VGF556‐576 was ineffective. VGF588‐617‐induced penile erection was reduced by nitroω‐l‐arginine methylester (L‐NAME; 20 µg), by morphine (5 µg) and by muscimol (1 µg), but not by dizocilpine [(+)MK‐801; 1 µg], nor by cis‐flupenthixol (10 µg) given into the paraventricular nucleus 10 min before the VGF peptide. d(CH2)5Tyr(Me)‐Orn8‐vasotocin (1 µg) effectively reduced VGF588‐617‐induced penile erection when given into the lateral ventricles but not when injected into the paraventricular nucleus. Immunocytochemistry with antibodies specific for the C‐terminal nonapeptide sequence of pro‐VGF (VGF609‐617) revealed numerous neuronal fibres and terminals within the paraventricular nucleus, including its parvocellular components. Here, many immunostained neuronal terminals impinged on parvocellular oxytocinergic neurons. The present results show for the first time that certain pro‐VGF C‐terminus‐derived peptides promote penile erection when injected into the paraventricular nucleus and suggest that, within this nucleus, these or closely related pro‐VGF‐derived peptides may be released to influence sexual function by activating paraventricular oxytocinergic neurons mediating penile erection.