Implication of the VGF-derived peptide TLQP-21 in mouse acute and chronic stress responses.

Razzoli M et al., Behav Brain Res. 2012 Apr 15;229(2):333-9. Epub 2012 Jan 24.

Implication of the VGF-derived peptide TLQP-21 in mouse acute and chronic stress responses.

The impact of stress is widely recognized in the etiology of multiple disorders. In particular, psychological stress may increase the risk of cardiovascular, metabolic, immune, and mood disorders. Several genes are considered potential candidates to account for the deleterious consequences of stress and recent data point to role of Vgf. VGF mRNA is abundantly expressed in the hypothalamus, where it has been involved in metabolism and energy homeostasis; more recently a link between VGF-derived peptides and mood disorders has been highlighted. The following experiments were performed to address the contribution of the VGF-system to stress induced changes in mice: the distribution of VGF immuno-reactivity in hypothalamic nuclei and its modulation by social stress; the role of VGF-derived peptide TLQP-21 in plasma catecholamine release induced by acute restraint stress (RS); the efficacy of chronic TLQP-21 in a mouse model of chronic subordination stress (CSS). VGF fibers were found in high density in arcuate, dorsomedial, and suprachiasmatic and, at lower density, in lateral, paraventricular, and ventromedial hypothalamic nuclei. Central administration of either 2 or 4mM TLQP-21 acutely altered the biphasic serum epinephrine release and decreased norepinephrine serum levels in response to RS. Finally, 28-day of 40μg/day TLQP-21 treatment increased CSS-induced social avoidance of an unfamiliar conspecific. Overall these data support a role for TLQP-21 in stress responses providing a promising starting point to further elucidate its role as a player in stress-related human pathologies.

Razzoli M et al., Behav Brain Res. 2012 Apr 15;229(2):333-9. Epub 2012 Jan 24.

Distribution of VGF peptides in the human cortex and their selective changes in Parkinson's and Alzheimer's diseases.

Cocco C et al., J Anat. 2010 Dec;217(6):683-93. doi: 10.1111/j.1469-7580.2010.01309.x. Epub 2010 Oct 12.

Distribution of VGF peptides in the human cortex and their selective changes in Parkinson's and Alzheimer's diseases.

VGF mRNA and its precursor-derived products are selectively expressed in certain neurons and promptly respond to neurotrophins and to neural/electrical activity. Proteomic studies have previously revealed a reduction in some VGF peptides in the cerebrospinal fluid of patients affected by Alzheimer's disease and other conditions, suggesting their potential diagnostic and clinical significance. As the presence of VGF peptides within the human cortex has been somewhat elucidated, they were studied postmortem in the frontal, parietal, and temporal cortex areas of control subjects and patients affected by Parkinson's disease, and in parietal cortex samples from patients with Alzheimer's disease. We raised antibodies to the C-/N-terminal portions of the proVGF precursor protein, to the TPGH and TLQP sequences and to the neuroendocrine regulatory peptide (NERP)-1, all used for enzyme-linked immunosorbent assay coupled with gel chromatography and for immunohistochemistry. In the control brain samples, the levels of TPGH and C-terminus peptides were about 130-200 and 700-2000 pmol g⁻¹, respectively, the N-terminus and NERP-1 peptides were less represented (about 10-30 and 4-20 pmol g⁻¹, respectively), and the TLQP peptides were below detection limits. Upon gel chromatography, the VGF antisera mainly revealed small molecular weight forms (i.e. about 0.8-1.3 kDa), whereas VGF immunolocalisation was found within different types of neuron in rat and bovine brain cortices. In the Parkinson's disease samples, a clear-cut decrease was revealed in the parietal cortex only, exclusively for TPGH and NERP-1 peptides, whereas in the Alzheimer's disease samples, a reduction in all of the VGF peptides was shown. The results suggest the involvement of VGF in the physiological or pathophysiological mechanisms occurring in the parietal cortex of patients with Parkinson's and Alzheimer's diseases.

Cocco C et al., J Anat. 2010 Dec;217(6):683-93. doi: 10.1111/j.1469-7580.2010.01309.x. Epub 2010 Oct 12.

 

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

Possenti R et al., Biochem J. 2011 Aug 31. [Epub ahead of print]

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 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. Here we provided a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We showed for the first time that VGF-ir is present within sympathetic fibers in white adipose tissue but not in adipocytes. Furthermore, we identified a saturable receptor binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in white adipose tissue as compared with other tissues, and selectively upregulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of norepinephrine/b adrenergic receptors pathways and dose-dependently decrease 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.

Possenti R et al., Biochem J. 2011 Aug 31. [Epub ahead of print]

 

Selective expression of TLQP-21 and other VGF peptides in gastric neuroendocrine cells and modulation by feeding.

Brancia C, et al. J Endocrinol. 2010 Dec; 207 (3): 329-41. Epub 2010 Sep 27

Selective expression of TLQP-21 and other VGF peptides in gastric neuroendocrine cells and modulation by feeding.

Although vgf gene knockout mice are hypermetabolic, administration of the VGF peptide TLQP-21 itself increased energy consumption. Agonist-antagonist roles are thus suggested for different VGF peptides, and the definition of their tissue heterogeneity is mandatory. We studied the rat stomach using antisera to C- or N-terminal sequences of known or predicted VGF peptides in immunohistochemistry and ELISA. TLQP (rat VGF(556-565)) peptide/s were most abundant (162±11 pmol/g, mean±s.e.m.) and were brightly immunostained in enterochromaffin-like (ECL) cells and somatostatin cells. A peptide co-eluting with TLQP-21 was revealed in HPLC of gastric and hypothalamic extracts, while the extended TLQP-62 form was restricted to the hypothalamus. Novel PGH (rat VGF(422-430)) peptide/s were revealed in ghrelin cells, mostly corresponding to low MW forms (0.8-1.5  kDa), while VGF C-terminus peptides were confined to neurons. VGF mRNA was present in the above gastric endocrine cell types, and was prominent in chief cells, in parallel with low-intensity staining for further cleaved products from the C-terminal region of VGF (HVLL peptides: VGF(605-614)). In swine stomach, a comparable profile of VGF peptides was revealed by immunohistochemistry. When fed and fasted rats were studied, a clear-cut, selective decrease on fasting was observed for TLQP peptides only (162±11 vs 74±5.3  pmol/g, fed versus fasted rats, mean±s.e.m., P<0.00001). In conclusion, specific VGF peptides appear to be widely represented in different gastric endocrine and other mucosal cell populations. The selective modulation of TLQP peptides suggests their involvement in peripheral neuro-endocrine mechanisms related to feeding responses and/or ECL cell regulation.

Brancia C, et al. J Endocrinol. 2010 Dec; 207 (3): 329-41. Epub 2010 Sep 27

In vitro and in vivo pharmacological role of TLQP-21, a VGF-derived peptide, in the regulation of rat gastric motor functions.

Severini, et al. Br J Pharmacol. Jul, 2009; 157(6):984-93. Epub 2009 May 19. PMID: 19466987

In vitro and in vivo pharmacological role of TLQP-21, a VGF-derived peptide, in the regulation of rat gastric motor functions.

BACKGROUND AND PURPOSE: Vgf gene expression has been detected in various endocrine and neuronal cells in the gastrointestinal tract. In this study we investigated the pharmacological activity of different VGF-derived peptides. Among these, TLQP-21, corresponding to the 556-576 fragment of the protein was the unique active peptide, and its pharmacological profile was further studied.
EXPERIMENTAL APPROACH: The effects of TLQP-21 were examined in vitro by smooth muscle contraction in isolated preparations from the rat gastrointestinal tract and, in vivo, by assessing gastric emptying in rats. Rat stomach tissues were also processed for immunohistochemical and biochemical characterization.
KEY RESULTS: In rat longitudinal forestomach strips, TLQP-21 (100 nmol x L(-1)-10 micromol x L(-1)) concentration-dependently induced muscle contraction (in female rats, EC(50) = 0.47 micromol.L(-1), E(max): 85.7 +/- 7.9 and in male rats, 0.87 micromol x L(-1), E(max): 33.4 +/- 5.3; n = 8), by release of prostaglandin (PG)E(2) and PGF(2a) from the mucosal layer. This effect was significantly antagonized by indomethacin and selective inhibitors of either cyclooxygenase-1 (S560) or cyclooxygenase-2 (NS398). Immunostaining and biochemical studies confirmed the presence of VGF in the gastric neuronal cells. TLQP-21, injected i.c.v. (2-32 nmol per rat), significantly decreased gastric emptying by about 40%. This effect was significantly (P < 0.05) blocked by i.c.v. injection of indomethacin, suggesting that, also in vivo, this peptide acts in the brain stimulating PG release.
CONCLUSIONS AND IMPLICATIONS: The present results demonstrate that this VGF-derived peptide plays a central and local role in the regulation of rat gastric motor functions.

Severini, et al. Br J Pharmacol. Jul, 2009; 157(6):984-93. Epub 2009 May 19. PMID: 19466987

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

Bartolomucci A, et al. Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14584-9.

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 2-AR (2 adrenergic receptor) and white adipose tissue (WAT) PPAR- (peroxisome proliferator-activated receptor ), 3-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.

Bartolomucci A, et al. Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14584-9.

 

VGF is required for obesity induced by diet, gold thioglucose treatment, and agouti and is differentially regulated in pro-opiomelanocortin- and neuropeptide Y-containing arcuate neurons in response to fasting

Hahm S., et al. The Journal of Neuroscience, August 15, 2002, 22(16):6929-6938

VGF is required for obesity induced by diet, gold thioglucose treatment, and agouti and is differentially regulated in pro-opiomelanocortin- and neuropeptide Y-containing arcuate neurons in response to fasting

Targeted deletion of the gene encoding the neuronal and neuroendocrine secreted polypeptide VGF (nonacronymic) produces a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA levels are regulated in the hypothalamic arcuate nucleus in response to fasting. To gain insight into the site(s) and mechanism(s) of action of VGF, we further characterized VGF expression in the hypothalamus. Double-label studies indicated that VGF and pro-opiomelanocortin were coexpressed in lateral arcuate neurons in the fed state, and that VGF expression was induced after fasting in medial arcuate neurons that synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in this region of the hypothalamus in fasted mice was inhibited by exogenous leptin. In leptin-deficient ob/ob and receptor-mutant db/db mice, VGF mRNA levels in the medial arcuate were elevated. To identify neural pathways that are functionally compromised by Vgf ablation, VGF mutant mice were crossed with obese A(y)/a (agouti) and ob/ob mice. VGF deficiency completely blocked the development of obesity in A(y)/a mice, whereas deletion of Vgf in ob/ob mice attenuated weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were dramatically elevated 10- to 15-fold above those of wild-type mice. VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF mutant mice to monosodium glutamate-induced obesity are consistent with a role for VGF in outflow pathways, downstream of hypothalamic and/or brainstem melanocortin 4 receptors, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis.

Schematic model indicating the sites where VGF may regulate energy balance, based on genetic, histochemical, and lesion data. Arcuate orexigenic and anorexigenic projections are shown as gray and white, respectively. Regions of the nervous system in which VGF mRNA or protein is abundantly expressed and/or regulated by feeding or GI manipulation are indicated by a speckled pattern. The dashed lines identify areas susceptible to GTG- or MSG-induced injury. Dark-gray projections outlined in black, downstream of agouti effects on the MC4-R, represent possible candidate circuits that might be regulated by locally synthesized VGF (speckled areas). ARC, Arcuate nucleus; DRG, dorsal root ganglia; LH, lateral hypothalamus; VMN, ventromedial nucleus.

 

 

VGF is required for the development of obesity in agouti mice. Representative agouti (Ay/a) and double-mutant (Vgf/Vgf,Ay/a) mice are shown (A). Body weight (B) and body composition (C) measurements for the indicated genotypes are shown (mean ± SE). Histograms identified by different letters are significantly different from one another (B) (p < 0.05; ANOVA with Tukey-Kramer post hoc comparisons; n = number of mice analyzed of the indicated genotype).

 

 

VGF is required for the development of hyperphagia but not increased adiposity in ob/ob mice. Representative males of the following genotypes are shown: VGF mutant (Vgf/Vgf), double-mutant (Vgf/Vgf,ob/ob), ob/ob, and wild type (A). Body weight (B), body composition (C), and daily food intake (D) were measured for the indicated genotypes (mean ± SE). Histograms identified by different letters are significantly different from one another (B, D) (p < 0.05; ANOVA with Tukey-Kramer post hoc comparisons; n = number of mice analyzed of the indicated genotype).

Hahm S., et al. The Journal of Neuroscience, August 15, 2002, 22(16):6929-6938

 

Characterization of the reproductive effects of the anorexigenic VGF-derived peptide, TLQP-21: In vivo and in vitro studies in male rats.

Pinilla., et al. Am J Physiol Endocrinol Metab., Feb 8, 2011, Epub ahead of print, PMID: 21304062

Characterization of the reproductive effects of the anorexigenic VGF-derived peptide, TLQP-21: In vivo and in vitro studies in male rats.

VGF (non-acronymic) is a 68 kDa protein, encoded by the homonymous gene, which is abundantly expressed at the hypothalamus and has been involved in the control of metabolism and body weight homeostasis. Different active peptide fragments are generated from VGF, including TLQP-21. Circumstantial evidence has suggested that VGF might participate also in the control of reproduction. Yet, its mechanisms of action and the eventual role of specific VGF-derived peptides on the hypothalamic-pituitary-gonadal (HPG) axis remain unknown. We report herein a series of studies on the reproductive effects of TLQP-21, as evaluated in male rats by a combination of in vivo and in vitro analyses. Central administration of TLQP-21 induced acute gonadotropin responses in pubertal and adult male rats, likely via stimulation of GnRH secretion, as documented by static incubations of hypothalamic tissue. In addition, in pubertal (but not adult) males, TLQP-21 stimulated LH secretion directly at the pituitary level. Repeated central administration of TLQP-21 to pubertal males subjected to chronic undernutrition was able to ameliorate the hypo-gonadotropic state induced by food deprivation. In contrast, chronic administration of TLQP-21 to fed males at puberty resulted in partial desensitization and puberty delay. Finally, in adult (but not pubertal) males, TLQP-21 enhanced hCG-stimulated testosterone secretion by testicular tissue in vitro. In sum, our data are the first to document a complex and multifaceted mode of action of TLQP-21 at different levels of the male HPG axis, with predominant stimulatory effects, therefore providing a tenable basis for the (direct) reproductive role of this VGF-derived peptide.

Pinilla., et al. Am J Physiol Endocrinol Metab., Feb 8, 2011, Epub ahead of print, PMID: 21304062