J Mol Endocrinol. 2015 Jun;54(3):227-39.

The VGF-derived peptide TLQP-62 modulates insulin secretion and glucose homeostasis.
 

Petrocchi-Passeri P1, Cero C2, Cutarelli A1, Frank C2, Severini C1, Bartolomucci A2, Possenti R3.
  • 1Department of Medicine of SystemsUniversity of Rome Tor Vergata, Via Montpellier, 100133 Rome, ItalyInstitute of Cell Biology and NeurobiologyCNR, Rome, ItalyDepartment of Integrative Biology and PhysiologyUniversity of Minnesota, Minneapolis, Minnesota, USAIstituto Superiore di SanitàRome, ItalyEBRI FoundationRome, Italy Department of Medicine of SystemsUniversity of Rome Tor Vergata, Via Montpellier, 100133 Rome, ItalyInstitute of Cell Biology and NeurobiologyCNR, Rome, ItalyDepartment of Integrative Biology and PhysiologyUniversity of Minnesota, Minneapolis, Minnesota, USAIstituto Superiore di SanitàRome, ItalyEBRI FoundationRome, Italy.
  • 2Department of Medicine of SystemsUniversity of Rome Tor Vergata, Via Montpellier, 100133 Rome, ItalyInstitute of Cell Biology and NeurobiologyCNR, Rome, ItalyDepartment of Integrative Biology and PhysiologyUniversity of Minnesota, Minneapolis, Minnesota, USAIstituto Superiore di SanitàRome, ItalyEBRI FoundationRome, Italy.
  • 3Department of Medicine of SystemsUniversity of Rome Tor Vergata, Via Montpellier, 100133 Rome, ItalyInstitute of Cell Biology and NeurobiologyCNR, Rome, ItalyDepartment of Integrative Biology and PhysiologyUniversity of Minnesota, Minneapolis, Minnesota, USAIstituto Superiore di SanitàRome, ItalyEBRI FoundationRome, Italy Department of Medicine of SystemsUniversity of Rome Tor Vergata, Via Montpellier, 100133 Rome, ItalyInstitute of Cell Biology and NeurobiologyCNR, Rome, ItalyDepartment of Integrative Biology and PhysiologyUniversity of Minnesota, Minneapolis, Minnesota, USAIstituto Superiore di SanitàRome, ItalyEBRI FoundationRome, Italy roberta.possenti@gmail.com.

 

Abstract

Insulin secretion control is critical for glucose homeostasis. Paracrine and autocrine molecules secreted by cells of the islet of Langerhans, as well as by intramural and autonomic neurons, control the release of different hormones that modulate insulin secretion. In pancreatic islets, the abundant presence of the granin protein VGF (nonacronymic; unrelated to VEGF) suggests that some of its proteolytically derived peptides could modulate hormone release. Thus, in the present study, we screened several VGF-derived peptides for their ability to induce insulin secretion, and we identified the VGF C-terminal peptide TLQP-62 as the most effective fragment. TLQP-62 induced a potent increase in basal insulin secretion as well as in glucose-stimulated insulin secretion in several insulinoma cell lines. We found that this peptide stimulated insulin release via increased intracellular calcium mobilization and fast expression of the insulin 1 gene. Moreover, the peripheral injection of TLQP-62 in mice improved glucose tolerance. Together, the present findings suggest that TLQP-62, acting as an endocrine, paracrine, or autocrine factor, can be considered a new, strong insulinotropic peptide that can be targeted for innovative antidiabetic drug discovery programs.

KEYWORDS: GSIS; diabetes; intracellular calcium; neuropeptide; signaling

PMID: 25917832

 

Supplement

The vgf gene (non-acronymic) was originally described as a NGF-inducible neurosecretory protein. The gene product is the proVGF protein, a member of the Chromogranin/Secretogranin family. It is composed of 617 amino acids in rats/mouse and 615 in human. Most of the Secretogranin proteins are involved in granulogenesis formation but there are evidences of their proteolitically processing leading to the formation of smaller fragments with specific biological activities (Reviewed in Bartolomucci et al 2011).

The VGF precursor protein, that migrates as a 90 kDa MW band in western blot analysis, is processed by the neuroendocrine-specific prohormone convertases PC1/3 and PC2, producing a number of VGF-derived peptides (identified with the first four aminoacids of the sequence followed by the numer of aminoacids), which are stored in dense core granules and secreted through the regulated pathway (Reviewed in Ferri et al 2011).

Different studies have demonstrated that VGF-derived peptides have different biological functions. Notably, VGF gene has been involved in metabolic regulation and some of its derived peptides modulate feeding behaviour as well as energy expenditure, reproduction and nociception. (Reviewed in Lewis et al 2015).

In the present study, we demonstrate that all the b-cell lines tested, as well the Langherans Islet, the neuronal autonomic afference nerves and the pancreatic ganglia, contain VGF precursor (90-80 kDa band) and low molecular peptides, suggesting a potential role of VGF-derived peptides in the control of islets function. Furthermore, we used pancreatic b-cell lines to assess the direct effects of VGF C-terminal derived peptides on insulinotropic function.

Among all peptides that we tested, the TLQP-62 is the most potent insulin secretagogue in presence of low glucose concentration in the media (Figure 1 panel A). In addition, the C-terminal VGF peptides, especially the TLQP-62 peptide, are released upon secretagogue stimuli (Figure 1 panel B).  Moreover, other VGF derived peptides such as TLQP-21, which induces a very weak insulin secretion and controversial activity in vivo (Stephens SB, et al 2012; Christiansen CB el al 2015) and internal fragments such as Nerp1 (human PESA-25 or rat LEGS-25) and Nerp2 (QAEA-38) (Moin AS, et al 2012;  Kim et al 2015), modulate pancreatic islet functions suggesting a complexes regulatory mechanisms for VGF-derived peptides. In this respect it must be noted that germline VGF knockout result in altered islet morphology and low insulin plasma level (Watson et al. 2005).

The VGF peptides stimulatory activity is mediated by increased cytosolic Ca2+ concentration, as we could demonstrate using FURA2AM. The peptide TLQP-62 potently increases Calcium mobilization, while TLQP-21 or AQEE-30 are less effective (Figure 1 panel C).

The activity of TLQP-62 on intracellular Ca2+ mobilization is affected by treatment with thapsigargin, which opens internal Ca2+ storage, depleting slowly and irreversibly the internal pool of Ca2+, but also by extracellular Ca2+ concentration, indicating strong Calcium induced Calcium release in insulinoma cells.

Although the receptor for TLQP-62 peptide has not been identified yet, the intracellular signalling indicates a modulation of PKC, ERK and AMPK pathways. In INS-1E cell line we demonstrate that High Glucose, PKA activation (via Forskoline) and TLQP-62 have a modulatory positive transcriptional control on Ins-1 and Vgf gene expression.

Finally, we demonstrate that acute injection of TLQP-62 (5mg/kg) in lean non-obese and non-diabetic mice, significantly improved glucose tolerance (figure 1 panel D). Further studies should be direct to evaluate the roles of this peptide in obese or diabetic mice.

 

 

Possenti Fig1Figure 1

Panel A: Insulin secretion upon secretagogue agents in insulinoma cell line INS1E. VGF derived peptides (TLQP-62, TLQP-21 and AQEE-30) used  10 mM.

Panel B: Intracellular (left panel) and secretory (right panel) westrn blot profile from INS1E cells, using antiserum raised against the C-terminal portion of VGFprotein.

Panel C: Calcium intracellular fluxes upon different stimuli in INS1E cells.

Panel D: Glucose Tolerance Test in lean non-obese and non-diabetic wild type mice.

 

 

Most recently, an alterated profile of VGF fragments has been identified in T2D patients and in Obese Mice (D’Amato F, et al 2015).

Taken together, these results show that classical insulin secretory stimuli are able to cause also secretion of mature processed forms of VGF-derived peptides, mostly identified as TLQP-62 form. Moreover, this peptide is able to stimulate and reinforce insulin secretion in a paracrine/autocrine manner.

 

 

Possenti Fig2Figure 2: Model for Insulin and VGF derived peptides secretion in insulinoma cells and the autocrine/paracrine effect of these peptides.

 

 

The discovery that new regulatory peptides for insulin secretion have been identified, opens more questions on control of islet function.

Although the receptors for some of these peptides have not been identified and characterized yet, our new findings suggest that TLQP-62 should be regarded as a new target for drug discovery programs focus on treatment of impaired insulin secretion in metabolic disorders such as in Diabetes and Obesity.

 

References 

  1. Bertolomucci A. et al. The extended granin family: structure, function, and biomedical implications. Endocr Rev 2011 Dec;32(6):755-97. doi: 10.1210/er.2010-0027. 
  2. Christiansen CB, et al. The VGF-Derived Neuropeptide TLQP-21 Shows No Impact on Hormone Secretion in the Isolated Perfused Rat Pancreas. Horm Metab Res 2015 Jun;47(7):537-43. doi: 10.1055/s-0034-1395615
  3. D’Amato F, et al. VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice. PLos One, 2015 Nov 12;10(11):e0142333. doi: 10.1371/journal.pone.0142333 
  4. Ferri GL et al. VGF: an inducible gene product, precursor of a diverse array of neuro-endocrine peptides and tissue-specific disease biomarkers. J Chem Neuroanat. 2011 Dec;42(4):249-61. doi: 10.1016/j.jchemneu.2011.05.007.
  5. Kim JW, et al. Chronic effects of neuroendocrine regulatory peptide (NERP-1 and -2) on insulin secretion and gene expression in pancreatic β-cells. Biochem Biophis Rec Commun.  2015 Feb 6;457(2):148-53. doi: 10.1016/j.bbrc.2014.12.067. 
  6. Lewis JE, et al. Neuroendocrine role of VGF. Front Endocrinol 2015 Feb 2;6:3. doi: 10.3389/fendo.2015.00003
  7. Moin AS, et al. Neuroendocrine regulatory peptide-2 stimulates glucose-induced insulin secretion in vivo and in vitro.  Biochem Biophys Res Commun. 2012 Nov 30;428(4):512-7. doi: 10.1016/j.bbrc.2012.10.073
  8. Stephens SB, et al. A VGF-derived peptide attenuates development of type 2 diabetes via enhancement of islet β-cell survival and function. Cell Metab. 2012 Jul 3;16(1):33-43. doi: 10.1016/j.cmet.2012.05.011
  9. Watson E, et al. VGF ablation blocks the development of hyperinsulinemia and hyperglycemia in several mouse models of obesity. Endocrinology. 2005 Dec;146(12):5151-63. doi: 10.1038/mp.2011.73.

 

 

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